# Patent

## Title
Voltage-dependent calcium channel beta subunit functional core

## Abstract

The present invention relates to crystallized forms of a voltage dependent calcium channel β subunit functional core, methods of producing and methods of utilizing the same.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Provisional Application Ser. No. 60/569,642, filed May 11, 2004, which is hereby incorporated by reference.


FIELD OF THE INVENTION
The present invention relates to crystallized, voltage-dependent calcium channel β subunit functional cores, methods of producing the same and methods of use thereof for identification of agents that inhibit or promote voltage dependent calcium channel acitivity. This invention further relates to an isolated polypeptide of a voltage-dependent calcium channel (VDCC) β subunit functional core comprising SH3 and guanylate kinase-like domains and methods of regulating voltage-dependent calcium channels.
BACKGROUND OF THE INVENTION
The passage of Ca2+ in a selective manner across the lipid bilayer of the cellular plasma membrane occurs by way of several protein families, one of them being voltage-dependent calcium channels (VDCC). These channels are multi-protein assemblies that serve as “switchboards” for the highly regulated Ca2+ signal. They communicate with a large number of cellular players involved in amplifying, terminating or modulating this critical pathway.
Two classes of assemblies gate the flow of Ca2+ in response to the voltage state of the plasma membrane. The structurally related but distinct classes are sensitive to low (CaV3) or higher (CaV1 or 2) relative voltage shifts. CaV1 or 2 subfamilies couple excitation of the cell to a variety of processes, depending on cell type, including contraction, secretion and transcription. Furthermore, signal pathway crosstalk requires regulation of channel action by a small host of molecules. An example of such interactions is the GPCR signaling modulation of CaV2 channels. Association of liberated Gbg with the calcium channel as a result of GPCR activation has a notable effect on function, with Gbg behaving as an allosteric effector.
The VDCC (CaV1 or 2), as defined by biochemical purification of the stable complex, comprises four distinct polypeptides: α1, α2d, β and γ. α1 is the membrane pore forming subunit, which contains four transmembrane domains repeats, paralleling the tetrameric architecture of potassium channels. Each domain contains the canonical voltage-dependent ion channel organization i.e. six putative transmembrane segments. The membrane domains are connected by linkers located in the intracellular milieu, as are both the amino- and carboxy-termini. β, in contrast, is a soluble and intracellular protein. Four separate β genes have been catalogued, each with multiple splice variants. All four genes are expressed in the brain, while other tissues exhibit essential gene-specific expression, giving rise to embryonic lethality in β1 and β2 knockout mice. β3 and β4 knockouts or alleles lead to pathologies in a variety of physiological systems.
Since molecular cloning of β, much attention has been focused on its role in VDCC function. Two major directions emerged from these studies. First, β facilitates the proper localization or trafficking of the VDCC and the α1 subunit in particular to the cellular plasma membrane. Several different β isoforms chaperone the channel to its target. Second, β acts as an important modulator of the channel's electrophysiological properties. β alters activation and inactivation kinetics, causes a leftward shift in the I-V curve and, on the single channel level, induces an increase in the channel opening probability. Furthermore, the localization functionality has a marked impact on the electrophysiological aspect by increasing the number of channels at the membrane, significantly enhancing current amplitude.
The groundwork for understanding β's molecular mechanism was laid with the discovery that β bound α1 through a region in the linker between domain I and II, labeled the AID (for α1 interaction domain). Later studies have shown that β also interacts with other regions of α1, depending on the isoform, but the AID appears to be the primary, high affinity site of interaction. Notably, structure-function research has implicated the AID itself in playing a critical role in channel activity, especially in terms of its effects on current inactivation.
Examination of β's structure and functional correlates has been more limited. Sequence analysis of the various genes from differing species supported a division of the protein into two central conserved motifs flanked by diverging sequences. Though it is thought that these domains interact in a stable fashion, and represent a functional core of the full-length β protein, a framework for understanding β's function in the context of the VDCC and any other possible roles is as yet, lacking.
SUMMARY OF INVENTION
In one embodiment, this invention provides a crystallized voltage-dependent calcium channel (VDCC) β subunit functional core, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the voltage-dependent calcium channel β functional core to a resolution of better than 5.0 Angstroms, characterized as:

    
    
        (a) Form I of a crystallized voltage-dependent calcium channel β functional core, wherein said crystal has a space group of P21212 , with unit cell dimensions of a=34.8, b=74.1, c=163.8 Å, and α=β=γ=90°; or
        (b) Form II of a crystallized voltage-dependent calcium channel β functional core, wherein said crystal has a space group of P41212 with unit cell dimensions of a=b=75.6, c=164.4 Å, and α=β=γ=90°.
    
    


In another embodiment, this invention provides a computer readable data storage material encoded with computer readable data comprising structure coordinates of Table 1.
In another embodiment, this invention provides a crystallized voltage-dependent calcium channel β functional core in complex with an AID peptide, wherein the AID peptide comprises the amino acid sequence corresponding to or homologous to SEQ ID NO: 22; wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of voltage-dependent calcium channel β functional core in complex with an AID peptide to a resolution of better than 5.0 Angstroms, wherein said crystal has a space group of P21212 with unit cell dimensions of a=77.8, b=168.3, c=34.2, Å, and α=β=γ=90°.
In another embodiment, this invention provides a method of using a crystal of this invention in an inhibitor screening assay comprising:

    
    
        (a) selecting a potential inhibitor by performing rational drug design with the three-dimensional structure determined for said crystal, wherein said selecting is performed in conjunction with computer modeling;
        (b) contacting said potential inhibitor with a voltage-dependent calcium channel; and
        (c) detecting the ability of said potential inhibitor for inhibiting said voltage-dependent calcium channel.
    
    


In another embodiment, this invention provides a method of growing a crystallized voltage-dependent calcium channel (VDCC) β subunit functional core, comprising growing the crystal by vapor diffusion using a reservoir buffer containing 1.6 M ammonium sulfate, 0.1M Hepes, pH=7.5, and 5 mM β-mercaptoethanol, at 4° C.
In another embodiment, this invention provides a method of growing a crystallized voltage-dependent calcium channel (VDCC) β subunit functional core, comprising growing the crystal by vapor diffusion using a reservoir buffer containing 3% PEG 20,000, 0.1M Bicine, pH=9, and 100 mM NaCl, and 5 mM β-mercaptoethanol, at 19° C.
In another embodiment, this invention provides a method of growing a co-crystallized voltage-dependent calcium channel (VDCC) β subunit functional core and an AID peptide, comprising:

    
    
        (a) contacting said voltage-dependent calcium channel (VDCC) β subunit functional core with an AID peptide comprising an amino acid sequence corresponding to or homologous to SEQ ID NO: 22, under conditions in which a voltage-dependent calcium channel (VDCC) β subunit functional core and AID peptide complex is formed; and
        (b) growing the crystal by vapor diffusion using a reservoir buffer containing 1-4% PEG 20,000; 0.1M Bicine, pH=9, and 1-3% MPD, and 5mM βmercaptoethanol, at 19° C.
    
    


In another embodiment, this invention provides a method for identifying a test compound that inhibits the interaction between a voltage-dependent calcium channel (VDCC) β subunit functional core and an AID peptide, said method comprising:

    
    
        (a) contacting a voltage-dependent calcium channel (VDCC) β subunit functional core or a peptide fragment thereof comprising the binding site for an AID peptide with an AID peptide, and a test compound, under conditions and for a time sufficient to permit the formation of a complex between said voltage-dependent calcium channel (VDCC) β subunit functional core or peptide fragment thereof and said AID peptide;
        (b) contacting a voltage-dependent calcium channel (VDCC) β subunit functional core or a peptide fragment thereof comprising the binding site for an AID peptide with an AID peptide, and no test compound, under conditions and for a time sufficient to permit the formation of a complex between said voltage-dependent calcium channel (VDCC) β subunit functional core or peptide fragment thereof and said AID peptide;
        (c) detecting the presence of a complex in (a) versus (b),

whereby a decrease or absence in the complex detected in (a) as compared to (b) indicates that the test compound inhibits the interaction between a voltage-dependent calcium channel (VDCC) β subunit functional core and an AID peptide.

    
    


According to this aspect of the invention, and in another embodiment, the binding site for an AID peptide comprises an M residue at position 246, 247 or combinations thereof. In another embodiment, the binding site for an AID peptide comprises an A residue at position 250 or an L residue at position 251, a V or L residue at position 343, an I or V residue at position 345, a K residue at position 349, a V residue at position 350, an R residue at position 353, an L or R residue at position 354, an S residue at position 357, an E residue at position 390, an N residue at position 391, an L residue at position 393, or an A residue at position 396, or combinations thereof, of the voltage-dependent calcium channel (VDCC) β subunit functional core.
In another embodiment, this invention provides an isolated polypeptide of a voltage-dependent calcium channel (VDCC) β subunit functional core comprising SH3 and guanylate kinase-like domains. According to this aspect of the invention, and in another embodiment, the polypeptide has an amino acid sequence that shares at least 90% identity with SEQ ID NO: 20 or 21. In another embodiment, the SH3 domain of the polypeptide comprises a Y residue at position 71, a W residue at position 105, or a P residue at position 121, or combinations thereof.
In another embodiment, this invention provides a method for promoting voltage-dependent calcium channel activity, comprising contacting a voltage-dependent calcium channel with an agent that initiates or enhances the physical organization of:

    
    
        (a) An AID peptide; and
        (b) Domain II of the β subunit of said voltage-dependent calcium channel;

whereby initiated or enhanced physical organization promotes channel activation, thereby promoting voltage-dependent calcium channel activity.

    
    


According to this aspect of the invention, and in another embodiment, the agent stimulates or increases the production of a negative electrostatic potential in Domain II of the β subunit, at a region contiguous with AID binding. In another embodiment, the β subunit changes its shape, length, rigidity, or combination thereof, in response to changes in electrostatic potential in said voltage-dependent calcium channel.
In another embodiment, this invention provides a method for inhibiting voltage-dependent calcium channel activity, comprising contacting a voltage-dependent calcium channel with an agent that interferes with the physical organization of:

    
    
        (a) An AID peptide; and
        (b) Domain II of the β subunit of said voltage-dependent calcium channel;

whereby interference by said agent prevents the physical organization promoting channel activation, thereby inhibiting voltage-dependent calcium channel activity.

    
    


According to this aspect of the invention, and in another embodiment, the agent interferes with production of a negative electrostatic potential in Domain II of the β subunit, at a region contiguous with AID binding, thereby inhibiting voltage-dependent calcium channel activity. In one embodiment, the agent constrains the N-terminus of said β subunit, or in another embodiment, the agent anchors said N-terminus in a cell membrane.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 is a schematic of the rabbit VDCC β2a primary sequence, a representative of the β family, and the various constructs prepared in this study. The lightly stippled box denotes the first conserved domain, domain I, and the darkly stippled box denotes the second conserved domain, domain II. The BID, a sequence responsible for binding to VDCC α1, is located as shown in the beginning of domain II (Opatowsky et al., (2003). J Biol Chem 278, 52323-52332).
FIG. 2 demonstrates the purification of recombinant, full-length VDCC β proteins. A: SDS-PAGE of VDCC β3 purification in its various stages; MW: Molecular weight markers (kD); lane 1: soluble fraction of the crude lysate; lane 2: metal chelate column flow through fraction; lane 3: metal chelate column eluate fraction (first major purification step); lane 4: ion-exchange column flow through fraction; lane 5: ion-exchange column eluate fraction (second major purification step); lane 6: protein after TEV digestion which removes a 26 residue amino terminus including the histidine tag, producing a distinct mobility shift; lane 7: hydroxylapatite column flow through fraction; lane 8: hydroxylapatite column eluate (final major purification step). B: SDS-PAGE of purified VDCC β2a; MW: Molecular weight markers (kD); neighboring lane: gel-filtration column eluate (final major purification step) after concentration (Opatowsky et al., 2003).
FIG. 3 demonstrates a limited proteolyses of full-length VDCC β proteins. A: β3 was incubated with activated papain at a ratio of 3000:1 on ice in 10 mM Tris pH 8, 200 mM NaCl, 5-10 mM βME. Aliquots were taken from the reaction at the indicated times, SDS sample buffer was added, samples were boiled and analyzed later by SDS-PAGE, as shown. B: β2a incubated with activated papain and analyzed as for β3. Arrows indicate the protease resistant fragments (Opatowsky et al., 2003).
FIG. 4 demonstrates the association of domains I and II. A: SDS-PAGE gel stained with Coomassie blue of a pull-down assay. Domain I contains a polyhistidine tag with which it was immobilized, while domain II was without a tag. Protein input is indicated by the key above the gel. Left lane: domain I pulled down with metal-chelate resin; center lane: domain II pulled down with metal-chelate resin (there is a small degree of non-specific binding to the resin); right lane: immobilized domain I after incubation with domain II for 20 minutes in phosphate buffer pH 7, containing 300 mM NaCl. Protein input was on the order of 10 mg protein. Beads were washed five times with buffer for all experiments. The output of domain II in the right lane is dramatically enhanced due to association with domain I. Relevant bands are labeled. B: Gel-filtration chromatography elution profile of domain I alone (dotted trace) and linkerless core (solid line), after digestion with TEV protease to remove the linker. The insets show SDS-PAGE gels of the relevant central fractions from each peak. As can be easily discerned, domain I and II coelute in the left-hand peak. Relevant bands are labeled (Opatowsky et al., 2003).
FIG. 5 demonstrates circular dichroism spectra of VDCC β proteins. Each curve is labeled as per the legend. The ordinate is molar ellipticity units (deg cm2 dmol-1) (Opatowsky et al., 2003).
FIG. 6 demonstrates binding isotherms of VDCC β with a fluorescein labeled AID peptide as measured by fluorescence polarization. A: full-length β2a gives a KD of 16.1±3.8 nM. B: linkerless core i.e. domain I plus domain II gives a KD of 6.6±1.3 nM. C: relative binding curves: filled circles are domain I alone; empty circles NusA-domain II fusion after incubation with TEV protease; filled triangles are domain I plus NusA-domain II fusion after incubation with TEV protease. Estimated protein concentration is in nM units (Opatowsky et al., 2003).
FIG. 7 demonstrates a functional assay of purified β proteins. Physiological activities of the purified proteins were examined by two-electrode voltage clamp analysis of oocytes expressing VDCC by combination of mRNA and protein injection. (A) VDCC consisted of α1.2 and β2 proteins. (B) VDCC consisted of α1.2 and β3 proteins. PB: protein buffer, Pro: Protein, DI: domain I, DI+II: domain I+domain II prepared by proteolysis of full length protein and subsequent purification (Opatowsky et al., 2003).
FIG. 8 demonstrates VDCC β protein family sequence conservation. 24 protein sequences of VDCC β were selected from the NCBI database, based on a search with Blast. Splice variants were removed and the selection included several invertebrate and vertebrate sequences. These sequences were run on the ConSeq server (http://conseq.bioinfo.tau.ac.il), which uses the Max4Site algorithm (Pupko, T., Bell, R. E., Mayrose, I., Glaser, F., and Ben-Tal, N. (2002) Bioinformatics 18 Suppl 1, S71-77) to compute sequence conservation scores. Scores account for evolutionary distances and are normalized to units of standard deviation. Zero represents the average evolutionary rate and less than 0 indicates increasing conservation. The scores were then averaged in a window of ±five residues (i.e. 11 total residues) for every residue and plotted as seen in the histogram. Arrows indicate the residue numbers of blocks of significant conservation. These blocks correspond quite well with the biochemically defined domains I and II. The reference sequence for numbering is rabbit β2a (Opatowsky et al., 2003).
FIG. 9 is a photograph of an SDS-PAGE of the fused core construct purification steps. The gel-filtration eluate was used for crystallization.
FIG. 10 is a photograph of rod shaped form I (A) and plate-like form II (B) crystals of voltage gated calcium channel beta subunit (fused core construct).
FIG. 11 demonstrates oscillation frames (0.5°) of form I (A) and form II (B) crystals. Diffraction data are observed to 3.4 Å and 2.3 Å, respectively
FIG. 12 demonstrates a ribbon (A) and topology (B) diagram of the VDCC β subunit functional core bound to the AID peptide Domain I. Domain II and the AID are represented in red, blue and green, respectively. The topology scheme for each domain was patterned and labeled after the relevant canonical motif.
FIG. 13 depicts sequence alignments and secondary structures for the VDCC β functional core (A) and the α1 subunit I-II linker until the end of the AID sequence (B). Sequence of rabbit CCβ2a (GenBank accession number CAA45575.1) (SEQ ID NO: 23), human CCβ3 (NP—000716) (SEQ ID NO: 24) and Anopheles gambiae CCβAnoph (EAA12209.1) (SEQ ID NO: 25) were aligned using CLUSTALW (A). For comparison, a structure-based sequence alignment was performed with mouse c-Crk, N-terminal SH3 domain (Q64010, PDB-1CKA) (SEQ ID NO: 26), S. cerevisiae guanylate kinase GUK yeast, (AAB64881, KIBYGU, PDB-1GKY) (SEQ ID NO: 27) and rat PSD-95 (NP—062567, PDB-1JXO) (SEQ ID NO: 28). Secondary structure elements were assigned with DSSPcont (Andersen et al., 2002), with nomenclature and color codes as in FIG. 12B. Arrows, cylinders, and flags denote β-strands, α-helixes, and 310 helixes, respectively. Highlighted in yellow are the residues involved in poly-proline recognition in canonical SH3 domains, represented by Crk. Residues highlighted in green participate in AID/β interactions. Boxed in cyan is the NMP-binding motif of guanylate kinase, as seen for yeast GUK and PSD-95. Arg131 of yeast GUK, essential for ATP binding, is highlighted in red. (B) Alignment of the α1 subunit I-II linker until the end of the AID sequence. Accession numbers are as follows: HUMAN Cav1.1 (Q13698) (SEQ ID NO: 29), Cav1.2 (Q13936) (SEQ ID NO: 30), Cav1.3 (Q01668) (SEQ ID NO: 31), Cav1.4 (060840) (SEQ ID NO: 32), Cav2.1 (000555) (SEQ ID NO: 33), Cav2.3 (Q15878) (SEQ ID NO: 34), Cav2.1 ANOPH (EAA07643.1) (SEQ ID NO: 35), Cav2.2 (EAA07643.1) (SEQ ID NO: 36), Cav1.1 DISOC Disc. ommata (P56698) (SEQ ID NO: 37), and Cav2.1 C.ELE C. elegans (AAB03158.4) (SEQ ID NO: 38).
FIG. 14 is a superposition of VDCC β functional core with c-Crk, SH3 domain, GuK and PSD-95. (A) Stereo diagram of β domain I (red), superimposed with PSD-95 SH3 domain (1JXO) (blue) and c-Crk SH3 domain (1CKA) (green) with a bound proline-rich peptide (yellow). (B) β domain II (blue) superimposed with GuK (yellow). β domain II was superimposed with yeast Guk (1GKY) (top). The same perspective was used to compare the lid and core subdomains of the GuK fold from the AID-β complex and mouse GuK with bound ADP (1LVG). (bottom). (C) AID-bound β (left) and PSD-95 (right) after superposition of β domain II and the PSD GuK domain. PSD was then translated horizontally. β domain I, domain II and AID are red, blue and green, respectively, while the PSD SH3 and GuK domains are cyan and yellow, respectively.
FIG. 15 demonstrates AID/β interactions. (A) 2Fo-Fc electron density map, contoured at 1σ, of the AID, calculated following molecular replacement using the β model and the AID/β complex data set. The AID (green) is represented as bonds and the β is represented in blue ribbons. (B) Closeup view of AID/β interactions with relevant side chains labeled. Dashed lines indicate hydrogen bonds. Helices 4.1 and 4.2 comprise the lid subdomain. (C) Table of AID mutations abstracted from the literature (Berrou et al., 2002; De Waard et al., 1996; Witcher et al., 1995). Binding to β is the effect charted on a scale of no binding (−) to WT binding (4 stars).
FIG. 16 demonstrates results of CD Spectroscopy of the AID peptide in solution. Spectra were measured in 2.5 mM Na phosphate pH=8, 15 mM NaCl (filled circles) at a peptide concentration of 46 μM at 20° C., and in 40% (vol/vol) trifluorethanol plus the above buffer (open circles). Deconvolution analysis (Bohm et al., 1992) indicate 9% helicity in buffer alone versus about 34% in the TFE solution.
FIG. 17 demonstrates structural features of VDCC β. (A) Proposed model for β localization, in respect to the α1 pore-forming unit. β is represented in a molecular surface form, domain I and domain II are red and blue, respectively. The I-II linker from S6 through the AID was built as a helix and is colored in green. (B) Electrostatic potential representation of the AID/β complex, projected onto its molecular surface. Potential was calculated at 0.1 M ionic strength. The corresponding worm representations are shown, where domain I, domain II and AID are red, blue and green, respectively. Note the negative patch in the proximity of the AID binding site.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides, in one embodiment crystallized voltage-dependent calcium channel β subunit functional cores, and methods of producing the same. This invention provides, in another embodiment, an isolated polypeptide of a voltage-dependent calcium channel (VDCC) β subunit functional core comprising SH3 and guanylate kinase-like domains. In another embodiment, this invention provides methods of regulating voltage-dependent calcium channels.
Calcium channels are membrane-spanning, multi-subunit proteins that allow controlled entry of Ca2+ ions into cells from the extracellular fluid. The most common type of calcium channel is voltage dependent. All “excitable” cells in animals, such as neurons of the central nervous system (CNS), peripheral nerve cells and muscle cells, including those of skeletal muscles, cardiac muscles, and venous and arterial smooth muscles, have voltage-dependent calcium channels.
Voltage dependent calcium channels allow for influx of Ca2+ ions into a cell, and require a depolarization to a certain level of the potential difference between the inside of the cell bearing the channel and the extracellular environment bathing the cell.
Calcium channels are present in various tissues and have a central role in regulating intracellular calcium ion concentrations, and are implicated in a number of vital processes in animals, including neurotransmitter release, muscle contraction, pacemaker activity, and secretion of hormones and other substances. These processes, are involved in numerous human disorders, such as CNS and cardiovascular diseases and therefore, methods of modulating functions of voltage-dependent calcium channels, which, in another embodiment, are provided in the present invention, may be utilized for treating, or alleviating symptoms of such human disorder, as will be described further, hereinbelow.
The VDCC comprises four distinct polypeptides: α1, α2d, β and γ. The β subunit interacts with the α subunit, and acts by both chaperoning VDCCs to the membrane and modulating gating of the channel.
This invention demonstrates, in one embodiment, that the β subunit is comprised of two motifs, which are homologous to the SH3 and the guanylate kinase families, respectively, and serve as a minimal requisite for MAGUK family members. The two domains were shown herein to interact in a stable fashion, and represent, in one embodiment, a functional core of the full-length β protein.
In one embodiment, the VDCC β subunit has a sequence such as that disclosed in Genbank Accession Number: AAH41811, AAH26479, AAQ97611, AAQ97610, AAQ97609, AAQ97608, AAQ97607, AAQ97606, AAQ97605, NP—666235, NP—000716, NP—000717, NP—660099, NP—446303, NP—059042, O00305, Q9Y698, P54284, Q08289, Q02641, Q9MZL7, Q9MZL5, Q9MZL3, AAL16951, AAL16950, AAL16949, AAL16948, I65767, I65766, I52859, I52859, P54288—2, or BAA07803.
Crystallographic analysis was conducted herein, of the VDCC β subunit functional core alone and in complex with an AID peptide. Three crystal forms were produced, as described hereinbelow.
In one embodiment, this invention provides a crystallized voltage-dependent calcium channel (VDCC) β subunit functional core, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the voltage-dependent calcium channel β functional core to a resolution of better than 5.0 Angstroms. In one embodiment, the crystallized voltage-dependent calcium channel (VDCC) β subunit functional core is characterized as having a space group of P21212, with unit cell dimensions of a=34.8, b=74.1, c=163.8 Å, and α=β=γ=90°.
In another embodiment, the crystallized voltage-dependent calcium channel (VDCC) β subunit functional core is characterized as having a space group of P41212 with unit cell dimensions of a=b=75.6, c=164.4 Å, and α=β=γ=90°.
In another embodiment, this invention provides a computer readable data storage material encoded with computer readable data comprising structure coordinates of Table 1.
In another embodiment, this invention provides a crystallized voltage-dependent calcium channel β functional core in complex with an AID peptide, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of voltage-dependent calcium channel β functional core in complex with an AID peptide to a resolution of better than 5.0 Angstroms. In one embodiment, the crystallized voltage-dependent calcium channel β functional core in complex with an AID peptide core is characterized as having a space group of P21212 with unit cell dimensions of a=72.8, b=168.3, c=34.2 Å, and α=β=γ=90°. In another embodiment, the AID peptide comprises the amino acid sequence corresponding to or homologous to QQLEEDLRGYMSWITQGE (SEQ ID NO: 22). In one embodiment, the AID peptide may comprise a substitution of A for Q at positions 1, 2 or combination thereof of SEQ ID NO: 22. In another embodiment, the AID peptide may comprise a substitution of S, D or R for an E at positions 4, 5 or combination thereof of SEQ ID NO: 22. In another embodiment, the AID may comprise a combination of any substitution described or exemplified herein.
In one embodiment, the AID peptide is as disclosed in Genbank Accession Numbers Q13698, Q13936, Q01668, O60840, O00555, Q15878, EAA07643.1, EAA07643.1, P56698, or AAB03158.4.
The structures of the voltage-dependent calcium channel (VDCC) β subunit functional core of the invention provide especially meaningful guidance for the development of drugs to target and promote or inhibit its interaction with alpha subunits, which would serve to regulate VDCC activity. Because calcium channels have a central role in regulating intracellular calcium ion concentrations, they play a role in a number of human disorders, such as CNS and cardiovascular diseases. Compounds useful for treating various cardiovascular diseases exert their beneficial effects, in one embodiment, by modulating functions of voltage-dependent calcium channels present in cardiac and/or vascular smooth muscle. In one embodiment, these compounds bind to calcium channels and block, or reduce the rate of, influx of Ca2+ into the cells in response to depolarization of the cell membrane. The identification of such inhibitors has important clinical application, and can be accomplished, in one embodiment, via the methods of this invention,
The three dimensional structures provided in an embodiment of this invention, allow an understanding of the interactions between the α and β subunits of the VDCC, which enables those of ordinary skill in the art to utilize rational mechanism-based and structure-based drug design technology to develop specific agents which promote, and/or inhibit VDCC activity, for use as novel drugs. The design of such agents may be accomplished in one embodiment, using molecular modeling, which may have important clinically therapeutic capabilities.
Crystallization of the β2α functional core provided for the generation of experimental electron density maps at 2.9 Å resolution, in one aspect of this invention, which enabled tracing of the complete molecule with two clearly defined domains. Refinement of the atomic model continued with a 2.3 Å data set. Diffraction data to 3.5 Å was obtained for an alternate crystal form, crystallized with two different forms of the functional core protein. Subsequent, co-crystallization of the β functional core with the CaV1.1 AID yielded diffraction data measured to a dmin of 2.2 Å. The structure revealed a two-domain construction, with dimensions 94×54×48 Å, and defined a new variant of the membrane associated guanylate kinase (MAGUK) protein family, which function as molecular scaffolds, using their various domains to create a web of protein-protein interactions at or near the cell membrane.
Structural analysis provided an understanding of how the two domains interact. Strand E of domain I is covalently linked by a short four residue turn to domain II, and in addition, other interactions include hydrogen bonds from domain II helix 5 made with the distal loop of domain I. Side chains from the ear lobe and a turn before strand 4 of domain II interact with strand E of domain I. The domain interface in β is buried much of its accessible surface area (1500 Å2).
The AID forms an α-helix that nestles into a groove on the protein, shaped by the juxtaposition of the lid subdomain and the core subdomain P-loop, helix 1, strand 5 and helix 5, and has two legs stapling it down in the groove. One leg consists of W369 and I370. W369 binds in a deep pit on the domain II surface and makes many interactions with domain II residues. The second leg consists of G365 and Y366. Y366 is buried completely, making van der Waals interactions with β. Concomitantly, its hydroxyl group makes bifurcated hydrogen-bonds to two water molecules. These, in turn, mediate hydrogen-bonds to β main chain carbonyl (389) or amine (345) groups. The balance of connections involve van der Waals or hydrophobic interactions. Importantly, interactions between AID and domain II are not localized to one region in sequence space of β. Three regions in sequence space contribute to the AID binding site, so that for proper binding of the AID, one requires almost the complete domain II.
The structure coordinates provided may also be used to solve the structure of VDCC β subunit functional core mutants, co-complexes with the same, or of the crystalline form of any other protein with significant amino acid sequence homology thereof.
One embodiment for a method that may be employed for such purposes is molecular replacement. In this method, the unknown crystal structure may be determined using the VDCC β subunit functional core structure coordinates of this invention.
In one embodiment, the term “molecular replacement” refers to a method that involves generating a preliminary model of a crystal of, in one embodiment, a channel protein, whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known, such as the VDCC β subunit functional core coordinates, within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal, as is known by those of ordinary skill in the art. Using the structure coordinates of VDCC β subunit functional core provided by this invention, molecular replacement can thus be used to determine the structure coordinates of, in other embodiments, a crystalline mutant or homologue of VDCC β subunit functional core, or additional crystal forms of VDCC β subunit functional core.
In another embodiment, this invention provides a method of using a crystal of this invention in an inhibitor screening assay comprising:

    
    
        (a) selecting a potential inhibitor by performing rational drug design with the three-dimensional structure determined for said crystal, wherein said selecting is performed in conjunction with computer modeling;
        (b) contacting said potential inhibitor with a voltage-dependent calcium channel; and
        (c) detecting the ability of said potential inhibitor for inhibiting said voltage-dependent calcium channel.
    
    


In one embodiment, the potential inhibitor is contacted with a VDCC β subunit functional core.
In another embodiment, this invention provides a method for identifying a test compound that inhibits the interaction between a voltage-dependent calcium channel (VDCC) β subunit and an AID peptide, said method comprising:

    
    
        (a) contacting a voltage-dependent calcium channel (VDCC) β subunit or a fragment thereof comprising the binding site for an AID peptide with an AID peptide, and a test compound, under conditions and for a time sufficient to permit the formation of a complex between said voltage-dependent calcium channel (VDCC) β subunit functional core or peptide fragment thereof and said AID peptide;
        (b) contacting a voltage-dependent calcium channel (VDCC) β subunit functional core or a peptide fragment thereof comprising the binding site for an AID peptide with an AID peptide, and no test compound, under conditions and for a time sufficient to permit the formation of a complex between said voltage-dependent calcium channel (VDCC) β subunit functional core or peptide fragment thereof and said AID peptide;
        (c) detecting the presence of a complex in (a) versus (b),

whereby a decrease or absence in the complex detected in (a) as compared to (b) indicates that the test compound inhibits the interaction between a voltage-dependent calcium channel (VDCC) β subunit functional core and an AID peptide.

    
    


According to this aspect of the invention, and in another embodiment, the binding site for an AID peptide comprises an M residue at position 246, 247 or combinations thereof. In another embodiment, the binding site for an AID peptide comprises an A residue at position 250 or an L residue at position 251, a V or L residue at position 343, an I or V residue at position 345, a K residue at position 349, a V residue at position 350, an R residue at position 353, an L or R residue at position 354, an S residue at position 357, an E residue at position 390, an N residue at position 391, an L residue at position 393, or an A residue at position 396, or combinations thereof, of the voltage-dependent calcium channel (VDCC) β subunit.
In another embodiment, according to this aspect of the invention, the method employs contacting the agent with a VDCC β subunit functional core.
In another embodiment, this invention provides a method for identifying a test compound that promotes the interaction between a voltage-dependent calcium channel (VDCC) β subunit and an AID peptide, said method comprising:

    
    
        (a) contacting a voltage-dependent calcium channel (VDCC) β subunit or a fragment thereof comprising the binding site for an AID peptide with an AID peptide, and a test compound, under conditions and for a time sufficient to permit the formation of a complex between said voltage-dependent calcium channel (VDCC) β subunit or peptide fragment thereof and said AID peptide;
        (b) contacting a voltage-dependent calcium channel (VDCC) β subunit or a peptide fragment thereof comprising the binding site for an AID peptide with an AID peptide, and no test compound, under conditions and for a time sufficient to permit the formation of a complex between said voltage-dependent calcium channel (VDCC) β subunit or peptide fragment thereof and said AID peptide;
        (c) detecting the presence of a complex in (a) versus (b),

whereby an increase in the amount of complex detected in (a) as compared to (b), or an increase in the binding affinity of said AID peptide for said VDCC β subunit in (a) as compared to (b) indicates that the test compound promotes the interaction between a voltage-dependent calcium channel (VDCC) β subunit functional core and an AID peptide.

    
    


According to this aspect of the invention, and in another embodiment, the binding site for an AID peptide comprises residues as indicated hereinabove. In another embodiment, according to this aspect of the invention, the method employs contacting the agent with a VDCC β subunit functional core.
Numerous computer programs are available and suitable for rational drug design and the processes of computer modeling, model building, and computationally identifying, selecting and evaluating potential inhibitors and promoters of VDCC α and β association in the methods described herein. These include, for example, GRID (available form Oxford University, UK), MCSS (available from Molecular Simulations Inc., Burlington, Mass.), AUTODOCK (available from Oxford Molecular Group), FLEX X (available from Tripos, St. Louis. Mo.), DOCK (available from University of California, San Francisco), CAVEAT (available from University of California, Berkeley), HOOK (available from Molecular Simulations Inc., Burlington, Mass.), and 3D database systems such as MACCS-3D (available from MDL Information Systems, San Leandro, Calif.), and UNITY (available from Tripos, St. Louis. Mo. Potential agents may also be computationally designed “de novo” using such software packages as LUDI (available from Biosym Technologies, San Diego, Calif.), LEGEND (available from Molecular Simulations Inc., Burlington, Mass.), and LEAPFROG (Tripos Associates, St. Louis, Mo.). Compound deformation energy and electrostatic repulsion, may be evaluated using programs such as GAUSSIAN 92, AMBER, QUANTA/CHARMM, AND INSIGHT II/DISCOVER. These computer evaluation and modeling techniques may be performed on any suitable hardware including for example, workstations available from Silicon Graphics, Sun Microsystems, and the like. These techniques, methods, hardware and software packages are representative and are not intended to be comprehensive listing. Other modeling techniques known in the art may also be employed in accordance with this invention. See for example, N. C. Cohen, Molecular Modeling in Drug Design, Academic Press (1996) (and references therein), and software identified at internet sites including the CAOS/CAMM Center Cheminformatics Suite at www.caos.kun.nl/, and the NIH Molecular Modeling Home Page at www.fi.muni.cz/usr/mejzlik/mirrors/molbio.info.nih.gov/modeling/software list/.
The agent is selected by performing rational drug design with the three-dimensional structure (or structures) determined for the crystal described herein, especially in conjunction with computer modeling and methods described above. The agent is then obtained from commercial sources or is synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. The agent is then assayed, in one embodiment, to determine its ability to promote or inhibit α and β association, or, in another embodiment, VDCC channel activity, by methods well known in the art, or, in another embodiment, as exemplified herein.
The agent selected or identified by the aforementioned process may be assayed to determine its ability to affect VDCC channel activity, in one embodiment. The assay may be in vitro or in vivo. The compounds described herein may be used in assays, including radiolabelled, antibody detection and fluorometric, in another embodiment, for the isolation, identification, or structural or functional characterization of VDCC. Such assays may include, in another embodiment, an assay, utilizing a full length VDCC, which, in another embodiment, is contacted with the agent and a measurement of the binding affinity of the agent against a standard is determined.
In one embodiment, the assay is performed as exemplified herein in Example 1. The assay may, according to this aspect of the invention, employ fluorescence polarization measurements. Agents, such as, in one embodiment, peptides which are expected to bind to the VDCC β subunit functional core are labeled with fluorescein. Labeled agent/peptide is then titrated with increasing concentrations of β, and the fluorescence polarization emitted by the labeled agent/peptide is determined. Fluorescence emission polarization is proportional to the rotational correlation time (tumbling) of the labeled molecule. Tumbling, in part, depends on the molecular volume, i.e. larger molecules have larger volume and slower tumbling which in turn gives rise to increased polarization of emitted light. If the agent/peptide associates with β, its effective molecular volume greatly increases, which may be evidenced by values obtained for polarization fluorescence emissions.
In other embodiments, electrophysiological methods for measuring calcium channel activity, which are known to those of skill in the art and exemplified herein may be utilized for the indicated purposes. Any such methods may be used in order to detect the formation of functional calcium channels and to characterize the kinetics and other characteristics of the resulting currents. Pharmacological studies may be combined with the electrophysiological measurements, in other embodiments, in order to further characterize the calcium channels
In one embodiment, complexes of peptides/agents with the VDCC β subunit may be studied using well-known X-ray diffraction techniques, and as exemplified herein, in another embodiment, and may be refined versus 2-3 angstrom resolution X-ray data to an R value of about 0.20 or less using readily available computer software, such as X-PLOR (Yale University©, 1992, distributed by Molecular Simulations, Inc.; Blundel & Johnson, 1985, specifically incorporated herein by reference).
The design of compounds that promote or inhibit VDCC α and β subunit interaction and channel activity, according to this invention may involve, in another embodiment, several considerations. In one embodiment, the compound should be capable of physically and structurally associating with the β subunit, such as, in other embodiments, by using non-covalent molecular interactions, including hydrogen bonding, van der Waals and hydrophobic interactions and the like. Second, the compound should be able to assume a conformation that allows it to associate with the VDCC β subunit. Although certain portions of the compound will not directly participate in this association with the VDCC β subunit, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., AID binding site with the VDCC β subunit.
The potential inhibitory or promotional effect of a chemical compound on the VDCC α and β subunit interaction and VDCC activity may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques, as is known to those of ordinary skill in the art.
One of ordinary skill in the art may use, in other embodiments of this invention, any one of several methods to screen chemical entities or fragments for their ability to associate with the VDCC β subunit, and, in another embodiment, with the AID binding site of the VDCC β subunit. This process may begin by visual inspection of, for example, the AID binding site of the VDCC β subunit on the computer screen based on data presented in, for example, FIG. 15. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the AID binding site of the VDCC β subunit. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include, in one embodiment, the programs GRID, MCSS, AUTODOCK and DOCK.
Once suitable chemical entities or fragments have been selected, they may, in another embodiment, be assembled into a single compound. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of VDCC. This may be followed, in another embodiment, by manual model building using software such as Quanta or Sybyl.
Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include, in other embodiments, CAVEAT, 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.) and HOOK.
In another embodiment, instead of proceeding to build an agent which interacts with the VDCC β subunit in a step-wise fashion, one fragment or chemical entity at a time as described above, the agent may be designed as a whole or “de novo” using an empty binding site. These methods may include the use of programs such as LUDI, LEGEND and LeapFrog, each of which represents an embodiment of this invention.
In another embodiment, once a compound has been designed or selected by the above methods, the efficiency with which that compound may bind to the VDCC β subunit may be tested and optimized by computational evaluation. In such methods, the deformation energy of binding may be considered and agents, which interact with the VDCC β subunit, may be designed with a particular deformation energy of binding, as will be understood by one of ordinary skill in the art.
A compound designed or selected as binding to the VDCC β subunit may, in another embodiment, be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the VDCC β subunit. Such non-complementary (e.g., electrostatic) interactions include, in other embodiments, repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the bound agent and the VDCC β subunit, make, in another embodiment, a neutral or favorable contribution to the enthalpy of binding.
Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction, and may include, in other embodiments, Gaussian 92, revision C (Frisch, Gaussian, Inc., Pittsburgh, Pa.,© 1992); AMBER, version 4.0 (Kollman, University of California at San Francisco, © 1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass., 1994); or Insight II/Discover (Biosysm Teclnologies Inc., San Diego, Calif., © 1994).
In another embodiment, once an agent binding to a VDCC β subunit has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. In one embodiment, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Such substituted chemical compounds may then be, in another embodiment, analyzed for efficiency of fit to the VDCC β subunit by the same computer methods described in detail, above.
In another embodiment, this invention provides a method for promoting voltage-dependent calcium channel activity, comprising contacting a voltage-dependent calcium channel with an agent that initiates or enhances the physical organization of:

    
    
        (a) An AID peptide; and
        (b) Domain II of the β subunit of said voltage-dependent calcium channel;

whereby initiated or enhanced physical organization promotes channel activation, thereby promoting voltage-dependent calcium channel activity.

    
    


According to this aspect of the invention, and in another embodiment, the agent stimulates or increases the production of a negative electrostatic potential in Domain II of the β subunit, at a region contiguous with AID binding. In another embodiment, the β subunit changes its shape, length, rigidity, or combination thereof, in response to changes in electrostatic potential in said voltage-dependent calcium channel.
The AID-β complex structure has been demonstrated herein to have AID and β almost seamlessly fuse into a united surface. The resulting electrostatic potential produced shows that β domain II has a patch of negative potential, coincident and contiguous to the locale of AID binding, which reacts to changes in the electrostatic potential of the surrounding environment as a result of membrane depolarization and the channel opening with its flux of Ca2+ ions diffusing from the channel mouth. Hence, the bound β reconfigures the electrostatics of the intracellular side of the channel.
In one embodiment, β orders a part of the channel physically connected to its gate, essentially changing its shape, or, in other embodiments, length, mechanical properties, such as rigidity, or a combination thereof.
In another embodiment of this invention, through β binding, the gate is now connected to a linker with much greater mass and depending on isoform constrained in its movement by its own independent attachment to the membrane.
Upon depolarization, the I-II linker/β negative patch may move in reaction to the changing electrostatic potential of α1, supplementing the radial torque on S6 from the voltage sensor. The presumed helical conformation will lend the requisite rigidity for any movements of the AID-β particle to be communicated to the gate. Also, the additional mass weighing on membrane domain I may destabilize the closed conformation of its S6. Thus, β's action on the channel state may be categorized, in another embodiment of this invention, as allosteric modulation.
Agents may be specifically designed, via methodology described hereinabove, to initiate or enhance the physical organization of the channel. In one embodiment, the agent promotes for greater or prolonged binding between β and the AID. In another embodiment, the agent promotes the generation of a greater or prolonged accumulation of a negative potential in β domain II, to promote channel opening. The design of such agents will be well known to one skilled in the art, and may comprise mutated β proteins, and other agents that achieve the desired result.
In one embodiment, the methods for promotion of VDCC activity may find clinical application in the treatment of Lambert-Eaton myasthenic syndrome (LEMS) seen in some small cell lung cancer patients. LEMS is a human autoimmune disorder that impairs neuromuscular transmission such that patients with this syndrome have a defect in the Ca2+ dependent quantal release of acetylcholine from motor nerve terminals. In this syndrome, patients develop antibodies (presurmably initiated by expression of the channel proteins in their small cell lung cancer) that react with voltage-gated calcium channel polypeptides which block depolarization-induced Ca2+ influx leading to the myasthenia. The methods/agents proposed in this invention may serve, according to this aspect, to promote appropriate physical organization, and/or inhibit antibody binding, in the case of LEMS, such that proper VDCC activity occurs.
In another embodiment, this invention provides a method for inhibiting voltage-dependent calcium channel activity, comprising contacting a voltage-dependent calcium channel with an agent that interferes with the physical organization of:

    
    
        (a) An AID peptide; and
        (b) Domain II of the β subunit of said voltage-dependent calcium channel;

whereby interference by said agent prevents the physical organization promoting channel activation, thereby inhibiting voltage-dependent calcium channel activity.

    
    


According to this aspect of the invention, and in another embodiment, the agent interferes with production of a negative electrostatic potential in Domain II of the β subunit, at a region contiguous with AID binding, thereby inhibiting voltage-dependent calcium channel activity. In one embodiment, the agent constrains the N-terminus of said β subunit, or in another embodiment, agent anchors said N-terminus in a cell membrane. In another embodiment, the agent provides steric hindrace such that the physical organization is diminished or absent. According to this aspect of the invention, the agent may be an antibody.
According to this aspect of the invention, and in one embodiment, channel inactivation may be via a hinged-lid mechanism, wherein the AID/β surface serves as the lid, the channel opens, Ca2+ accumulates at the mouth, and attracts the lid by Coulombic forces and in coordination with Ca2+-dependent inactivation determinants of the α1 C-terminus.
Subsequently, residues in the AID that are solvent exposed i.e. those not involved in binding β and other residues of the linker find their receptor site possibly in the channel mouth, thereby blocking ion flow and inactivating the channel. According to this inactivation mechanism, the hinge, which enables the lid to swivel may be located at the link between S6 and the I-II linker i.e. the conserved glycine sequence at its start, and agents targeting this region may be involved, in one embodiment, in regulating VDCC channel function
In another embodiment, the slowing or inactivation of β's movement may be constrained through its N-terminus, since, according to this aspect of the invention, the “lid” would then not be able to move as easily towards the channel mouth. This notion then explains the slow inactivation of β2a, whose N-terminus is anchored in the membrane by palmitoylation. When the palmitoylation site is mutated, inactivation parameters resemble that of the other isoforms and splice variants.
In one embodiment, the methods of this invention, wherein blocking calcium transport would be of therapeutic value, which may be accomplished via the methods of this invention, may include osteosclerosis, stroke, head trauma, epilepsy, and chronic, neuropathic and acute pain. Calcium transport, especially that associated with N-type channels, is also implicated in other neurological disorders such as migraine, epilepsy, mood disorders, schizophrenia, and certain degenerative disorders. Other conditions that benefit from reduced calcium flux include depression, anxiety, and other psychoses. Cardiovascular conditions benefited include hypertension and cardiac arrhythmias. In another embodiment, inactivation of VDCC's may be beneficial in applications in developing pesticides.
In certain embodiments, the assays for identifying compounds that modulate calcium channel activity are practiced by measuring the calcium channel activity of a eukaryotic cell having a heterologous, functional calcium channel when such cell is exposed to a solution containing the test compound and a calcium channel selective ion and comparing the measured calcium channel activity to the calcium channel activity of the same cell or a substantially identical control cell in a solution not containing the test compound. The cell is maintained, in one embodiment, in a solution having a concentration of calcium channel selective ions sufficient to provide an inward current when the channels open. Methods for practicing such assays are known to those of skill in the art. For example, for similar methods applied with Xenopus laevis oocytes and acetylcholine receptors, see, Mishina et al. [(1985) Nature 313:364] and, with such ooocytes and sodium channels [see, Noda et al. (1986) Nature 322:826-828]; For similar studies which have been carried out with the acetylcholine receptor, see, e.g., Claudio et al. [(1987) Science 238:1688-1694].
The assays thus use cells that express functional calcium channels and measure functionally, such as electrophysiologically, the ability of a test compound to potentiate, antagonize or otherwise modulate the magnitude and duration of the flow of calcium channel selective ions, such as Ca++ or Ba++, through the heterologous functional channel. The amount of current, which flows through the recombinant calcium channels of a cell may be determined, in one embodiment, directly, such as electrophysiologically, or, in another embodiment, by monitoring an independent reaction which occurs intracellularly and which is directly influenced in a calcium (or other) ion dependent manner.
Any method for assessing the activity of a calcium channel may be used in conjunction with the methods described herein. For example, in one embodiment of the method for testing a compound for its ability to modulate calcium channel activity, the amount of current is measured by its modulation of a reaction which is sensitive to calcium channel selective ions and uses a eukaryotic cell which expresses a heterologous calcium channel and also contains a transcriptional control element operatively linked for expression to a structural gene that encodes an indicator protein. The transcriptional control element used for transcription of the indicator gene is responsive in the cell to a calcium channel selective ion, such as Ca2+ and Ba2+. The details of such transcriptional based assays are described, for example, in PCT International Patent Application No. PCT/US91/5625.
In another embodiment, this invention provides a method of growing a crystallized voltage-dependent calcium channel (VDCC) β subunit functional core, comprising growing the crystal by vapor diffusion using a reservoir buffer containing 1.6 M ammonium sulfate, 0.1M Hepes, pH=7.5, and 5 mM β-mercaptoethanol, at 4° C.
In another embodiment, this invention provides a method of growing a crystallized voltage-dependent calcium channel (VDCC) β subunit functional core, comprising growing the crystal by vapor diffusion using a reservoir buffer containing 3% PEG 20,000, 0.1M Bicine, pH=9, and 100 mM NaCl, and 5 mM β-mercaptoethanol, at 19° C.
In another embodiment, this invention provides a method of growing a co-crystallized voltage-dependent calcium channel (VDCC) β subunit functional core and an AID peptide, comprising:

    
    
        (a) contacting said voltage-dependent calcium channel (VDCC) β subunit functional core with an AID peptide comprising an amino acid sequence corresponding to SEQ ID NO: 22, under conditions in which a voltage-dependent calcium channel (VDCC) β subunit functional core and AID peptide complex is formed; and
        (b) growing the crystal by vapor diffusion using a reservoir buffer containing 1-4% PEG 20,000; 0.1M Bicine, pH=9, and 1-3% MPD, and 5 mM β-mercaptoethanol, at 19° C.
    
    


In another embodiment, this invention provides an isolated polypeptide of a voltage-dependent calcium channel (VDCC) β subunit functional core comprising SH3 and guanylate kinase-like domains. In one embodiment, the polypeptide will have an amino acid sequence, which corresponds to, or is homologous to SEQ ID NO: 20 or 21.
In one embodiment, the polypeptides of this invention include, but are not limited to, fragments of native polypeptides from any animal species (including as humans), and derivatives of native (human and non-human) polypeptides and their fragments, provided that they have a biological activity in common with a respective native polypeptide. “Fragments” comprise, in one embodiment, regions within the sequence of a mature native polypeptide. The term “derived” is meant to include, in another embodiment, amino acid sequence and glycosylation variants, and covalent modifications of a native polypeptide, whereas the term “variant” refers to amino acid sequence and glycosylation variants within this definition VDCC β subunit functional core-derived peptides include all peptides having a qualitative biological activity in common with a native VDCC β subunit functional core, comprising both SH3 and guanylate kinase-like domains, or in another embodiment, their consensus sequences, and may, according to additional embodiments of the invention, comprise the entire consensus sequence, or a polypeptide fragment thereof.
The term “peptide”, when in reference to any peptide of this invention, is meant to include, in other embodiments, native peptides (either, degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminal, C terminal or peptide bond modification, including, but not limited to, backbone modifications, and residue modification, each of which represents an additional embodiment of the invention. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).
It is to be understood that any VDCC β subunit functional core-derived peptide of the present invention may be isolated, generated synthetically, obtained via translation of sequences subjected to any mutagenesis technique, as well as obtained via protein evolution techniques, well known to those skilled in the art.
In one embodiment, the VDCC β subunit functional core-derived peptide of the present invention may be employed in the following applications: 1) screening assays; 2) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and 3) methods of treatment (e.g., therapeutic and prophylactic), as has been described hereinabove.
In one embodiment, the terms “homology”, “homologue” or “homologous”, refer to an amino acid sequence, which exhibits, in one embodiment at least 70% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 72% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 75% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 80% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 82% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 85% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 87% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 90% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 92% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 95% or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 97% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits at least 99% correspondence with the indicated sequence. In another embodiment, the amino acid sequence exhibits 95%-100% correspondence with the indicated sequence. Similarly, as used herein, the reference to a correspondence to a particular sequence includes both direct correspondence, as well as homology to that sequence as herein defined.
Homology, as used herein, may refer to sequence identity, or may refer to structural identity, or functional identity. By using the term “homology” and other like forms, it is to be understood that any molecule, that functions similarly, and/or contains sequence identity, and/or is conserved structurally so that it approximates the reference sequence, is to be considered as part of this invention.
According to this aspect of the invention, and in another embodiment, the SH3 domain comprises a Y residue at position 71, a W residue at position 105, or a P residue at position 121, or combinations thereof.
In one embodiment, the molecular architecture of the β functional core supports, and in another embodiment, maximizes protein-protein interactions with other proteins. Proteins that associate with β may comprise, in one embodiment, members of the small G-protein subfamily, Gem, Rad, and Rem and HP1g, involved in gene silencing. According to this aspect of the invenion, β may serve as a nexus of the signaling pathways tied to calcium and may therefore be an effective target for multiple clinically relevant scenarios.
The following examples are presented in order to more fully illustrate some embodiments of the invention They should, in no way be construed, however, as limiting the scope of the invention.
EXAMPLES
Example 1
The Voltage-Dependent Calcium Channel β Subunit Contains Two Stable Interacting Domains
Materials and Methods
Subcloning, Expression and Purifications of the VDCC β3 Subunit:
Recombinant rat VDCC β 3 subunit (M88751) was expressed in E. coli strain BL-21 Tuner (Novagen), containing the “RIL” Codon PlusTM plasmid (Stratagene), using a modified pET21 (Novagen) vector (an 8×His and TEV protease site were engineered between the pET21d's NcoI and BamHI sites—a gift of Dr. Sean Juo). The protein was purified by sequential metal-chelate, ion-exchange and hydroxylapatite chromatography.
Polymerase chain reaction (PCR) was used to engineer EcoRI and BamHI restriction sites into the β 3 gene. The oligonucleotide primers used for amplification of the β 3 gene from the original plasmid were the following: Sense primer A, 5′-GCGCGGATCCTATGACGACTCCTACGTCCCC (SEQ ID NO: 1); Antisense primer B, 5′-GCGCGGATCCTATGACGACTCCTACGTGCCC (SEQ ID NO: 2). PCR product was ligated into doubly digested (EcoRI and BamHI) pET21d vector. Positive clones were identified by restriction analysis and subsequently sequenced.
Transformed Tuner cells were grown for 3-4 hr. at 37° C. in 10 liters of LB media, containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Upon reaching an A600 of 0.3, the temperature was lowered to 16° C. and growth continued until the culture reached an A600 of 0.6. Protein expression was induced with 200 μM IPTG. Cells were harvested after 14 hr by centrifugation, frozen and suspended in 100 ml lysis buffer, buffer L (300 mM NaCl; 50 mM Na-phosphate, pH=7; 1 mM PMSF; 5 mM MgCl2; 0.2% Triton; 1 mM βME; 10 mg lysozyme; 1 mg DNase). After lysis by French pressure cell (Aminco), cell debris was removed by centrifugation at 20,000×g. The soluble fraction was loaded onto a pre-equilibrated metal chelate “Talon” (Clontech) column (buffer A—300 mM NaCl; 50 mM Na-phosphate, pH=7), at a flow rate of 1.5 ml/min. The column was washed with buffer A, containing 5 mM imidazole, until a stable base line was achieved. After elution with buffer A, supplemented with 150 mM imidazole, the protein eluate was then diluted 3 fold with water and loaded onto a pre-equilibrated Source-Q (Amersham Pharmacia) column (buffer Q—70 mM NaCl; 20 mM Na-phosphate, pH=7). The column was then washed with buffer Q and fractions were eluted with a shallow linear gradient of buffer Q, containing 70-300 mM NaCl. VDCC β3 containing fractions were pooled (130-150 mM NaCl) and diluted 2 fold in 600 mM NaCl to buffer H concentrations (10 mM phosphate buffer, pH=7; 300 mM NaCl) and subjected to TEV protease prepared in house. The proteolysis continued for 12 hr. Subsequently, the sample was loaded onto a pre-equilibrated (with H buffer) hydroxylapatite (Calbiochem) column and eluted with a linear gradient of H buffer 10-100 mM K-phosphate pH=7 (eluted at 50 mM K-phosphate). Pooled fractions were concentrated to 10 mg/ml using spin concentrators (Vivascience), divided into aliquots and flash-frozen in liquid N2.
Subcloning, Expression and Purification of VDCC β 2a Subunit
PCR was used to engineer EcoRI and BamHI restriction sites into the VDCC β 2a (X64297) gene. The oligonucleotide primers used for amplification of the new β 2a gene from the original plasmid were the following: Sense primer C, 5′-GCGCGGATCCCTTGACAGGCACCTCGCGGC-3′ (SEQ ID NO: 3); Antisense primer D, 5′-CGCCGAATTCTCATTGGCGGATGTA-3′ (SEQ ID NO: 4). Subsequent subcloning was as described for VDCC β 3.
The VDCC β 2a subunit was expressed as for β 3. Purification was as follows. The soluble fraction of the crude extract was loaded on a pre-equilibrated Ni-CAM column (Sigma) (buffer B—50 mM Na-phosphate, pH=8; 300 mM NaCl) and washed with buffer B, containing 5 mM imidazole. The protein was then eluted with buffer B, containing 150 mM imidazole, and diluted 6 fold, followed by loading onto a pre-equilibrated Q-sepharose (Amersham Pharmacia) column buffer C-20 mM Na-phosphate, pH=8; 40 mM NaCl; 5 mM βME). Fractions were eluted with a shallow linear gradient of buffer C containing 40-200 mM NaCl and subjected to TEV proteolysis for 12 hr. VDCC β 2a containing fractions were pooled (80-100 mM NaCl) and loaded onto a pre-equilibrated gel filtration column (buffer G—10 mM Tris pH=8; 10 mM βME; 200 mM NaCl). The protein was eluted with buffer G. The pooled fractions were further processed as for β 3.
Subcloning, Expression and Purification of VDCC β 2a Domain I
PCR was used to engineer EcoRI and BamHI restriction sites into the β 2a domain I construct. The oligonucleotide primers used for amplification of the β 2a domain I construct from the original β 2a plasmid were the following: sense primer E, 5′-GCGCGGATCCAGCCGTCCATCCGATTCAGATGTG-3′ (SEQ ID NO: 5); antisense primer F, 5′GCGCGAATTCTCACTTTGCTCTCTGTTCATGCTGTAG-3′ (SEQ ID NO: 6). Subsequent subcloning was as described for VDCC β 3.
The β 2a domain I was expressed as for β 3. Purification was as follows. The soluble fraction of the crude extract was loaded on a pre-equilibrated Talon column (buffer A) and washed with buffer A, containing 75 mM imidazole. The protein was then eluted with buffer A, containing 150 mM imidazole, pooled and subjected to TEV proteolysis for 12 hr. The protein was then diluted 3 fold with 300 mM NaCl, loaded on a pre-equilibrated (buffer H) hydroxylapatite column and eluted with a linear gradient of H buffer 10-140 mM K-phosphate (eluted at 70 mM K-phosphate). The pooled fractions, with the addition of 10 mM βME, were loaded onto a pre-equilibrated gel filtration column (buffer G) and eluted with buffer G. The pooled fractions were processed as above.
Subcloning, Expression and Purification of VDCC β 2a Domain II
PCR was used to engineer EcoRI and BamHI restriction sites into the β 2a domain II construct. The oligonucleotide primers used for amplification of the β 2a domain II construct from the original β 2a plasmid were the following: primer G, sense 5′-GCGCGGATCCCACTCCAAAGAGAAAAGAATGCCC-3′ (SEQ ID NO: 7); Antisense primer I, 5′-GCGCGAATTCTCAAAGGAGAGGGTTGGGGAGATTGCT-3′ (SEQ ID NO: 8). PCR product was ligated into a doubly digested (EcoRI and BamHI) pET43.1a (Novagen) plasmid, thereby encoding a C-terminal fusion to the NusA protein with a 6×His Tag and thrombin site separating the two. A modified pET43 (8×His followed by a TEV cleavage sequence was inserted between the pET43 SpeI and BamHI sites) was also prepared to express domain II in the same way as with the commercial pET43.
The β 2a domain II was expressed as above. Purification of the NusA-Domain II fusion proceeded as follows. The soluble fraction of the crude extract was loaded on a pre-equilibrated Ni-CAM column (buffer B) and washed with buffer B, containing 5 mM imidazole. The protein was then eluted with buffer B, containing 30 mM imidazole, loaded on a pre-equilibrated (H buffer) hydroxylapatite column and eluted with a linear gradient of buffer H 10-250 mM K-phosphate (eluted at 100 mM K-phosphate). The protein was then subjected to thrombin (Sigma) proteolysis (5 U/mg of fusion protein) for 14 hr. The cleaved protein was diluted 2 fold and applied to a pre-equilibrated Q-sepharose column with PBS. The flow-through fractions were pooled, divided into aliquots and flash-frozen in liquid N2.
Subcloning, Expression and Purification of VDCC β2a Removable Linker Core
Subcloning was stepwise. First, pre-digested PCR product encoding domain I was ligated into EcoRI and BamHI digested pET21d plasmid. Next, pET21d-domain I plasmid was prepared and digested with EcoRI and NotI for ligation with a pre-digested PCR product encoding domain II. Finally, PCR product, encoding the linker between domains I and II and two TEV proteolysis sites at both ends, was singly digested with EcoRI and ligated into pre-digested EcoRI vector, now containing both domain I and domain II Positive clones containing the insert in the correct orientation were identified using restriction analysis and sequencing.
PCR was used to engineer EcoRI, BamHI and NotI restriction sites into the β 2a domain I and domain II encoding fragments. The oligonucleotide primers used for amplification of the β 2a domain I fragment from the original β 2a plasmid were the following: sense primer E-5′ listed above; Antisense primer J, 5′-CGCGGAATTCCTTTGCTCTCTGTTCATGCTGTAG-3′ (SEQ ID NO: 9). The oligonucleotide primers used for amplification of the β 2a domain II fragment from the original β 2a plasmid were the following: Sense primer K-5′-CGGAATTCAAGCTTCACTCCAAAGAGAAAAGAATGCCC-3′ (SEQ ID NO: 10); Antisense primer L-5′ -TTATACTAGCGGCCGCTCAAAGGAGAGGGTTGGGGAGATT-3′ (SEQ ID NO: 11). The oligonucleotide primers used for amplification of the β2a linker fragment with the addition of two TEV sites from the original β2a plasmid were the following: sense primer M-5′-CGCGGAATTCGAAAACCTGTACTTTCAGGGCCAAGGGAAATTCTACT CCA-3′ (SEQ ID NO: 12); Antisense primer N-5′ CGCGGAATTCGCCCTGAAAGTACAGGTTTTCGGGTGACGTTACACTGT TT-3′ (SEQ ID NO: 13)
The VDCC β2a removable linker core was expressed as above. The soluble fraction of the crude extract was loaded onto a pre-equilibrated Ni-CAM column (buffer B) and washed with buffer B, containing 5 mM imidazole. The protein was then eluted with buffer B, containing 150 mM imidazole, pooled and diluted two fold, followed by loading onto a pre-equilibrated hydroxylapatite column (H buffer). Protein was eluted with a linear gradient of H buffer (10-250 mM K-phosphate) at about 120 mM K-phosphate. Pooled fractions were diluted 4 fold and subjected for 12 hr to TEV protease. 10 mM PME was added to the sample and loaded onto a pre-equilibrated gel filtration column (G buffer). Protein was eluted with buffer G. The pooled fractions were processed as above.
Limited Proteolysis of VDCC β2a and β3 Subunits
Papain (20 μg/ml) (Sigma-Aldrich) was activated for 30 min in activation buffer (1.25 mM EDTA; 6.25 mM cysteine; 62.5 mM βME, at pH=7), and added to 3 mg/ml VDCC β 3 or b2a in a 1:20 dilution. The final ratio of papain to β protein was 1:3000. Trypsin (60 μg/ml) was added to 3 mg/ml β 3 in a 1:20 dilution, giving a final ratio of protease to protein of 1:1000. Reactions were performed on ice. Proteolysis progress (at different time intervals) was monitored by SDS-PAGE. Proteolysis products were purified for further analysis by HPLC reverse phase chromatography using a C4 column (Vydac) with a shallow acetonitrile gradient 30-80% (both solvents were supplemented with 0.05% trifluoracetic acid).
AID Peptide Binding Assays
Fluorescence polarization was used to determine the equilibrium dissociation constant (KD) for the interaction between a fluorescein-labeled AID peptide, purified and various VDCC β constructs. The synthetic peptide was purified by HPLC reverse chromatography using a C18 column (Vydac) with a shallow acetonitrile gradient 20-80% (both solvents were supplemented with 0.05% trifluoracetic acid). Its sequence is derived from the AID motif of: the CaV1.1 I-II linker and is as follows: fluorescein-GGQQLEEDLRGYNSWITQGE-COOH (SEQ ID NO: 14). A mutant peptide, fluorescein-GGQQLEEDLRGSNSWITQGE-COOH (SEQ ID NO: 15) was prepared, in addition. Increasing concentrations of protein were incubated with 0.5 nM labeled peptide for 10 min in the dark at room temperature. Polarization measurements were taken with an ISS K2 fluorescence spectrophotometer at excitation and emission wavelengths of 492 and 520 nm, respectively, at 20° C., maintained by a temperature-controlled water bath. Polarization measurements were made with integration times on the order of 20 seconds, achieving a standard deviation of 5 percent of signal. Binding isotherms for the various samples were measured three independent times. Binding data were analyzed in SigmaPlot (SPSS) by nonlinear regression used to fit a binding function as defined by the following equation:




  
    Δ
    ⁢
    
      
    
    ⁢
    P
  
  =
  
    
      
        B
        max
      
      ⁢
      X
    
    
      
        K
        D
      
      +
      X
    
  




where X is the concentration of free ligand, ΔP is the change in fluorescence polarization of the fluorophore (baseline polarization of the labeled peptide alone was subtracted), Bmax is the maximum change in polarization upon saturation and KD is the concentration of ligand required to reach half maximal binding.
CD Spectroscopy
All CD measurements were performed with an Aviv CD spectrometer model 202. Spectra were measured over the range of 280-180 nm at a scan rate of 1 nm/sec. For all measurements, a cell with 0.1 mm path length was used. Each spectrum is an average of 4 scans. The raw data were corrected by subtracting the contribution of the buffer to the CD signal. Data were smoothed and converted to molar ellipticity units. The measurements were taken at a constant temperature of 20° C., with an approximate protein concentration of 35 μM. More precise concentration of protein was obtained using the predicted extinction coefficient of the proteins and their 280 nm absorbance. The proteins' molar extinction coefficients (M−1 cm−1) at 280 nm are: β 3—52300; β 2a—39760; β 2a linkerless core—24870. The difference spectrum (full length β 2a—linkerless core) was calculated according to (Ausio, J., Toumadje, A., McParland, R., Becker, R. R., Johnson, W. C., Jr., and van Holde, K. E. (1987) Biochemistry 26, 975-982). Deconvolution calculations were computed with CDNN (Bohm, G., Muhr, R., and Jaenicke, R. (1992) Protein Eng 5, 191-195) using the 33 data basis set.
Electrophysiology
Preparation of Xenopus laevis oocytes, injection of mRNA of VDCC subunits, and electrophysiological recording and analysis were performed, as described (Kang, M. G., Chen, C. C., Felix, R., Letts, V. A., Frankel, W. N., Mori, Y., and Campbell, K. P. (2001) J Biol Chem 276, 32917-32924). The negative control group were oocytes expressing CaV1.2 (α1C) subunit only by mRNA injection followed by injection of protein buffer (PB). The positive control group were oocytes expressing α1C and β3 subunits by mRNA injection, followed by injection of protein buffer (PB). The oocytes of experimental groups were injected with α1C mRNA followed by injection of VDCC β proteins, two days after the mRNA injection. Injection of protein (0.4 mg of protein per oocyte) was performed similarly as mRNA injection. Oocytes injected with VDCC β proteins were incubated one more day and the expressed calcium channel currents were recorded using the two-electrode voltage-clamp technique.
Results
Using a T7 expression system, an E. coli BL-21 derivative cell line that allows for fine-tuning IPTG induction by better controlling IPTG concentration, and low temperature growths, overexpression of soluble, full-length rat VDCC β3 was obtained. In addition, the rabbit β2a isoform was subcloned and expressed in the same system. The expression vectors encoded a histidine tag on the amino terminus, followed by a TEV protease site and the ensuing desired coding sequence. VDCC β2a manifested robust expression.
Purification schemes for the VDCC β isoforms all utilized as a first step subsequent to lysis, metal chelate resin chromatography to isolate the His-tagged target proteins. This first step usually produced protein that was greater than 80-85% homogeneous. Histidine tags were removed by cleavage with TEV protease. The TEV protease is highly specific and does not cleave other sites on the protein. Full length VDCC β3 was highly sensitive to proteolysis. Therefore, efficient and rapid chromatographic processing proved essential and was applied systematically. The full-length proteins are shown in FIG. 1.
An important and classical method of probing protein structure is limited proteolysis, which when applied to the determination of domain structure, assumes that flexible and exposed regions of the protein are available to the protease and thus subject to hydrolysis.
Both β2a and β3 recombinant full-length proteins were subjected to limited proteolysis using both papain, a highly non-specific protease, and trypsin, a more specific protease. The time course results for limited digestion by papain are in FIG. 2. Early in the time course, a stable polypeptide emerged which was an approximately 40 kD fragment. Subsequently, this fragment was further digested such that after 16 hours, 2 two stable fragments remained, one of higher mobility and another of medium mobility. Both isoforms basically followed the same pattern (β2a seems to have an intermediate fragment).
Preparative limited proteolyses of VDCC β3 were performed, and samples were separated and analyzed by mass spectrometry and amino-terminal peptide sequencing. Electrospray mass spectrometry produced masses of 13,198 and 23,938 Dalton for the two stable fragments. N-terminal sequencing of these fragments indicated that the smaller one primarily began at residue 23 whereas the larger fragment began at residue 158. Combining these data allowed us to demarcate the boundaries of these stable domains. The smaller fragment is defined from 23 to 138 of the β3 sequence and designated domain I while the larger fragment is defined from 158 to 371 and designated domain II. The β2a domain definitions are 25 to 146 and 204 to 423 for domains I and II, respectively, as determined by sequence alignment to the β3 boundaries
MALDI-MS analyses of the early proteolytic 40 kD fragment gave a cluster of masses centered around 39,420 Dalton. This mass is consistent with a fragment extending from residue 21 to 370, and fits as well with the mass spectrometry and peptide sequencing results of the stable domains. Moreover, the stable domains are derivatives of this early fragment as discerned by the time course results, requiring them to be equal or smaller than the early fragment. The large fragment was therefore a core protein.
Further support for the structural division, i.e. two domains with a flexible connecting linker, came from limited proteolysis of a recombinant protein, whose ends were engineered based on homology of the VDCC β family (see FIG. 3; predicted core).
In order to determine whether the two domains interact, constructs expressing His-tagged β2a domain I, alone, and domain II as a C-terminal fusion protein with NusA were prepared (FIG. 3). His-tagged domain I was mixed with domain II which had been isolated away from the fusion partner NusA and shown to bind in a pull-down assay, indicating stable association of the two domains (FIG. 4a). Further support for stable association of the two derives from chromatographic experiments, where gel filtration analysis of domain I alone gives an elution volume of 93 ml on a Superdex 200 size-exclusion column. When purified removable linker core protein (FIG. 3), was digested by TEV so that the domains were no longer covalently linked, to give linkerless core, and then run out on the identical column, both domains I and II coeluted at an elution volume of 83 ml (FIG. 4b). The significant shift in elution volume for domain I and coelution of both domains strongly supports association of domains I and II, with solution conditions, ruling out the presence of non-specific or adventitious association.
CD spectra of several VDCC β forms were measured, and are shown in FIG. 5. The spectra of the full-length β3 and β2a proteins are similar, indicating comparable secondary structure. Furthermore, these spectra indicate that the proteins are of a mixed alpha helix/beta sheet type, as seen by visual inspection of the curves as well as by deconvolution calculations. The calculations point to approximately 35 percent sheet and 15 percent helix. A comparison of β3 and the β2a linkerless core spectra shows a very strong resemblance between these two proteins. In addition, the difference spectrum between β2a full length protein and the β2a linkerless core protein gives insight into the segments of protein outside of domains I and II, namely the N-terminal segment, the linker between domains and the C-terminal segment. This spectrum points to a significant fraction of random coil structure as evidenced by the shift in minimum and magnitude towards 198 nm and diminution of the other minimum at 222 nm, consonant with the limited proteolysis results.
In order to determine structure—function correlations regarding domain analysis, and to have a robust in vitro assay for further correlations with structural and electrophysiological experiments, a novel assay that measures binding of VDCC β proteins to an α1 I-II linker-derived AID peptide was developed. The assay employed fluorescence polarization measurements. AID peptides of 18-20 residue length were synthesized, some of them labeled with fluorescein attached at the amino-terminus. Labeled peptide was then titrated with increasing concentrations of β, and the fluorescence polarization emitted by the labeled peptide was determined. Fluorescence emission polarization is proportional to the rotational correlation time (tumbling) of the labeled molecule. Tumbling, in part, depends on the molecular volume, i.e. larger molecules have larger volume and slower tumbling which in turn gives rise to increased polarization of emitted light. If the peptide associates with β, its effective molecular volume greatly increases, as evidenced by values of polarization fluorescence emissions. Equilibrium isotherm titrations were performed with full-length β2a and linkerless core β2a. The binding curves (FIGS. 6a and 6b) indicate high affinity single site binding on the order of 6-15 nM. The binding is specific since addition of unlabeled AID peptide lowers the polarization to basal levels, i.e. it effectively competes with the labeled peptide (data not shown)
The assay was further validated by testing a mutant AID peptide, which has a single amino acid change (Y to S, which has been shown to have less than five percent binding capability of WT. No binding of the mutant AID was detected with the assay, at concentrations of up to 350 nM β2a protein, though WT protein at this concentration reached saturation binding.
The binding assay was then used to determine which domains of β are required for association with the AID peptide (FIG. 6c). Binding of the β2a domain I was assessed, which was negligible in the estimated concentration regime tested. The NusA-domain II fusion, which contained a TEV protease site separating NusA and domain II was incubated with TEV protease and the labeled peptide for one hour and then fluorescence polarization measured, where domain II demonstrated significant binding affinity for the AID. Incubation of the β2a domain I protein and the NusA-domain II fusion protein, containing a TEV protease site separating NusA and domain II, with TEV protease and the labeled peptide demonstrated that the presumed domain I-domain II complex binds with even higher affinity. Since identical amounts of the various proteins were taken in the three stages of this experiment, the curves provided a relative measure of binding for the different domains and their individual contributions. Thus, domain I does not bind by itself to the AID at high affinity whereas domain II does. However, it is clear that domain I somehow contributes to the increased binding of the AID for the presumed domain I-domain II complex.
In order to determine that the bacterially expressed, recombinant proteins were physiologically active, two-electrode, two-voltage-clamp measurements of Xenopus oocytes expressing α1 and microinjected with our protein samples was performed. Increased current amplitude and a shift of the steady-state activation was determined in I-V plots of Cav1.2 currents (FIG. 7), by injection of β proteins (except domain I), demonstrating the functional activity of the β proteins. Data values and statistical analyses are described in Table 1. The current amplitude increase and change of steady-state activation are statistically significant in all experimental groups except a group injected with β2a domain I protein.







TABLE 1





Electrophysiological Parameters of the Cav1.2 oocyte expression


plus protein microinjections



















Properties
α1.2
α1.2 + β3 mRNA
α1.2 + Full β3 Pro.
α1.2 + Core β3 Pro.










Current amplitude (A)











I at 20 mV
 −0.15 ± 0.01
 −0.56 ± 0.08**
 −0.47 ± 0.04***
 −0.45 ± 0.04***


I at 30 mV
 −0.16 ± 0.01
 −0.48 ± 0.07**
 −0.44 ± 0.03***
 −0.41 ± 0.03***


n
10
11
10
8







Steady-state activation parameters from I-V curve











V1/2 (mV)
  22.60 ± 4.22
  10.67 ± 1.95**
  10.03 ± 3.36*
  10.65 ± 4.46*


k (mV)
  11.91 ± 0.84
   9.08 ± 0.36**
  10.56 ± 0.42
  10.06 ± 1.15


n
10
11
10
8






α1.2 + Full β2
α1.2 + Core β2
α1.2 + d1 + d2 β2


Properties
Pro.
Pro.
Pro.
α1.2 + d1 β2 Pro.










Current amplitude (A)











I at 20 mV
 −0.71 ± 0.09**
 −0.87 ± 0.09***
 −0.44 ± 0.04***
 −0.16 ± 0.01


I at 30 mV
 −0.49 ± 0.06**
 −0.70 ± 0.07***
 −0.34 ± 0.03**
 −0.18 ± 0.02


n
11
11
12
9







Steady-state activation parameters from I-V curve











V1/2 (mV)
   3.68 ± 0.33***
   6.84 ± 0.83***
   3.24 ± 0.93***
  24.00 ± 3.01


k (mV)
   8.48 ± 0.53***
   8.43 ± 0.23***
   9.49 ± 0.92
  12.18 ± 0.49


n
11
11
12
9





Values are presented as mean ± SEM.


*p < 0.05,


**p < 0.01, and


***p < 0.001 (respect to aI.2),


I: current,


V: potential,


V1/2: membrane potential for half-maximal activation,


k: slope factor,


n: cell number,


Pro.: Protein,


d: domain.






Furthermore, the results show that the core protein electrophysiological activity corresponds well to full-length protein activity. In addition, the injection of purified domain I and domain II proteins prepared by proteolysis of full length protein and subsequent purification, comparable to β2a linkerless core protein from our in vitro experiments, still enabled significant changes in the current amplitude and activation shift in a qualitatively similar manner as native protein. It should be noted that the injected recombinant β2a proteins were found subsequently to contain a mutation (P122R) due to the PCR subcloning. This mutation had no effect on the proteins' electrophysiological activity.
Example 2
Crystallization of the VDCC β Subunit Functional Core
Materials and Methods
Subcloning, Expression and Purification
The VDCC β2a linkerless core construct was subcloned into a modified pET21-d vector and expressed as previously described (Opatowsky, Y., Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A. (2003). J Biol Chem 278, 52323-52332). A VDCC β2a fused core construct i.e. domain I fused to domain II was prepared as follows. Subcloning was sequential PCR was used to engineer BamHI and EcoRI restriction sites into the β2a domain I encoding gene. Primers used were the following: sense, 5′-GCGCGGATCCAGCCGTCCATCCGATTCAGATGTG-3′ (SEQ ID NO: 16); antisense, 5′-CGCGGAATTCCTTTGCTCTCTGTTCATGCTGTAG-3′ (SEQ ID NO: 17). The PCR product was ligated into a doubly digested (BamHI, EcoRI) pET21-d vector. Next, NotI and EcoRI sites were introduced into the domain II fragment by PCR using the sense primer, 5′-CGGAATTCAAGCTTCACTCCAAAGAGAAAAGAATGCCC-3′ (SEQ ID NO: 18) and the antisense primer, 5′-TTATACTAGCGGCCGCTCAAAGGAGAGGGTTGGGGAGATT-3′ (SEQ ID NO: 19). Finally, the domain II PCR product was ligated into a doubly digested (EcoRI NotI) pET21-d vector, already containing the domain I encoding fragment. Positive clones were identified by restriction analysis and sequencing. The resulting gene encodes domain I fused to domain II with four residues (EFKL); SEQ ID No: 39) between them that arise due to the cloning procedure.
Expression of the proteins was essentially as described previously (Opatowsky et al., 2003, supra). In brief, protein was expressed in transformed Tuner (Novagen) E. coli grown in 2xYT media, containing 100 μg/ml ampicillin and 34 μg/ml of chloramphenicol at 16° C. for 14 hours. Cells were then harvested by centrifugation and frozen for subsequent use. Cell paste was suspended in 100 ml lysis buffer (300 mM NaCl; 50 mM NaPO4, pH 8; 1 mg DNase). After lysis by French press, cell debris was removed by centrifugation at 20,000×g. The soluble fraction was loaded onto a pre-equilibrated metal-chelate Ni-CAM (Sigma) column (buffer A: 300 mM NaCl; 50 mM NaPO4, pH 8) and washed with buffer A supplemented with 7 mM imidazole until a stable baseline was achieved. The protein was eluted with buffer A supplemented with 150 mM imidazole, diluted six fold with 10% glycerol, and loaded onto a Q-Sepharose (Amersham Pharmacia) column, pre-equilibrated with buffer B (70 mM NaCl; 20 mM NaPO4, pH 8). Fractions were eluted with a shallow gradient of buffer C (400 mM NaCl; 20 mM NaPO4, pH 8).
VDCC β2a containing fractions (180-220 mM NaCl) were pooled and subjected to TEV protease digestion, in order to remove the 8×His tag. Proteolysis continued for 12 hours, and subsequently the protein was loaded onto a hydroxylapatite (Calbiochem) column, pre-equilibrated with buffer D (200 mM NaCl; 50 mM NaPO4, pH 8), washed with buffer D until a flat baseline was achieved. Then, the protein was eluted with buffer E (200 mM NaCl; 200 mM NaPO4, pH 8; 5 mM β-mercaptoethanol), and applied to a pre-equilibrated Superdex-200 gel-filtration column (Amersham Pharmacia) with buffer F (200 mM NaCl; 20 mM Tris, pH 8; 5 mM β-mercaptoethanol). The elution peak was concentrated to 12 mg/ml using spin concentrators (Vivascience), divided into aliquots and flash frozen in liquid N2.
Crystallization
Initial screens of both proteins were performed at 4° C. and 19° C. with Hampton Screen I and II (Hampton Research) in 96-well sitting-drop plates (Corning). Drop size was 2 μl, with a sample-reservoir ratio of 1:1. After 24 hours, microcrystals and crystalline hits appeared with many high molecular weight PEG and ammonium-sulfate conditions. Crystallization was refined using hanging-drop vapor diffusion plates, by varying different precipitant concentrations versus different pHs and buffers. The addition of low concentrations of alcohols and salts, both at 4° C. and 19° C., gave optimal growth conditions for both the fused core and linkerless core forms. Both protein types gave thick rod shaped crystals at 1.6 M ammonium sulfate; 0.1 M Hepes, pH 7; 5 mM β-mercaptoethanol, grown at 4° C. (crystal form I). Crystals appeared after several hours and diffracted for 48 hours post setup. The fused core protein also gave plate shape crystals at three percent PEG 20,000 (Fluka); 0.1 M Bicine, pH 9; 100 mM NaCl, 5 mM β-mercaptoethanol, grown at 19° C. (crystal form II). These crystals appeared after 12 hours and diffracted for no longer than 36 hours post set up.
Data Collection
Prior to flash freezing in cryo-loops, crystals were gradually transferred to cryoprotecting solutions, containing, in addition to cryoprotectant (30% sucrose for the ammonium sulfate crystals, and 35% glycerol for the PEG crystals), all of the mother-liqueur components. Heavy atom soak crystals were prepared by adding a final concentration of 1 mM heavy atom solution to the mother liqueur, for 5 min prior to the cryoprotectant soak. Flash frozen crystals were then placed on the 110° K N2-cooled goniometer head for data collection. Using home x-ray source (rotating anode), crystals were screened for diffraction quality. Synchrotron data collection was conducted at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The diffraction data were processed with DENZO/SCALEPACK (Otwinowski and Minor, 1997).
Results
Functional cores of the voltage-dependent calcium channel β subunit (β2a isoform), with an amino acid sequence corresponding to SRPSDSDVSL EEDREAVRRE AERQAQAQLE KAKTKPVAFA VRTNVSYSAA HEDDVPVPGM AISFEAKDFL HVKEKFNNDW WIGRLVKEGC EIGFIPSPVK LENMRLQHEQ RAK EFKL HSKEKRMPFF KKTEHTPPYD VVPSMRPVVL VGPSLKGYEV TDMMQKALFD FLKHRFEGRI SITRVTADIS LAKRSVLNNP SKHAIIERSN TRSSLAEVQS EIERIFELAR TLQLVVLDAD TINHPAQLSK TSLAPIVVYV KISSPKVLQR LIKSRGKSQA KHLNVQMVAA DKLAQCPPEL FDVILDENQL, EDACEHLADY LEAYWKATHP PSSNLPNPLL* (SEQ ID NO: 20), and SRPSDSDVSL EEDREAVRRE. AERQAQAQLE KAKTKPVAFA VRTNVSYSAA HEDDVPVPGM AISFEAKDFL HVKEKFNNDW WIGRLVKEGC EIGFIPSRVK LENMRLQHEQ RAK EFKL HSKEKRMPFF KKTEHTPPYD VVPSMRPVVL VGPSLKGYEV TDMMQKALFD FLKHRFEGRI SITRVTADIS LAKRSVLNNP SKHAIIERSN TRSSLAEVQS EIERIFELAR TLQLVVLDAD TINHPAQLSK TSLAPIVVYV KISSPKVLQR LIKSRGKSQA KHLNVQMVAA DKLAQCPPEL FDVILDENQL EDACEHLADY LEAYWKATHP PSSNLPNPLL* (SEQ ID NO: 21), respectively, were expressed in E. coli, and purified to homogeneity (FIG. 9). The functional cores were crystallized, and two crystal forms predominated (FIG. 10). While crystal form II (dmin=2.3 Å) (panel B) should provide us with a detailed atomic structure, form I (dmin=3.6 Å) (panel A) may provide structural information about conformational variability of these two-domain molecules.
To date, attempts to obtain experimental phasing by isomorphous replacement have been thwarted, since crystal form II suffers from significant non-isomorphism, as evident by the large deviations in the unit cell size along the longest axis and by the high c2 values (>20) obtained by scaling independent data sets, including native versus native sets (FIG. 11). Additional data processing statistics for the voltage gated calcium channel β subunit crystals are provided in Table 2.







TABLE 2







Properties of the crystals of VDCC β subunit functional core:










Protein
Fused core
Fused core
Linkerless core





Crystal form
Form II
Form I
Form I


Wavelength (Å)
  0.933
  0.976
  0.976


Space group
P21212
P41212
P41212


Unit-cell
a = 34.8
a = b = 75.6
a = b = 76.5


Parameters (Å)
b = 74.1
c = 164.4
c = 164.9



c = 163.8
α = β = γ = 90°
α = β = γ = 90°



α =



β = γ = 90°


Total reflections
71732
25341
46242


Unique reflections
18271
 6853
 6448


Completeness (%)
92.8 (87.9)
96.5 (98.1)
 100 (100)


Rmerge (%)
 4.8 (31)
 5.3 (49)
  10 (43)


I/σ
15.4 (4.3)
19.5 (2)
13.2 (4)


Resolution range
50-2.3
50-3.4
50-3.55


(Å)


Beamline
ID-14-2
BM-14
BM-14





**Values in parentheses are for the highest resolution shells. Data were collected at the ESRF, Grenoble, France






Example 3
Structural Determination of the VDCC β Subunit
Materials and Methods
Expression and Purification
Selenomethionine-substituted VDCC β functional core was prepared and crystallized for multiwavelength anomalous diffraction (MAD). Protein was produced in E. coli BL21 (DE3) as described in Example 1, by inhibition of the methionine pathway (Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1993). J Mol Biol 229, 105-124). An overnight starter culture was grown from a single transformed colony in 10% LB medium. LB media was removed prior to the introduction of 2 L of New Minimal Media (Budisa, N., Steipe, B., Demange, P., Eckerskorn, C., Kellermann, J., and Huber, R. (1995). High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. Eur J Biochem 230, 788-796), fortified with Kao and Michayluk vitamin solution (Sigma), and 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Cells were grown at 37° C. to OD600=0.3, whereupon the temperature was lowered to 16° C. Lysine, phenylalanine and threonine (100 mg/L), isoleucine, leucine and valine (50 mg/L) and DL-selenomethionine (50 mg/L) were added 45 min before induction (A600=0.6). Expression was induced with 200 μM IPTG over a 14 hr period. Purification of the SeMet protein was similar to that of the native protein (Example 2), except that 5 mM β-ME was added to all solutions to prevent oxidation. The efficiency of SeMet incorporation was confirmed by mass spectrometry.
Crystallization and Structure Determination
SeMet protein crystals were grown at 19° C. by hanging drop vapor diffusion with conditions near those of native protein (Example 1). Equal volumes (1-2 μl) of diluted frozen stock protein (6-12 mg/ml) were mixed with reservoir solution containing 1-4% PEG 20K, 0.1 M Bicine pH 9, 1-3% MPD and 5 mM β-mercaptoethanol. Orthorhombic crystals were allowed to grow for no longer than 20 hr, then cryoprotected by sequential dilutions with mother liquor added with 35% glycerol. The crystals were mounted in cryoloops and flash frozen with liquid N2.
For co-crystallization of VDCC β functional core and purified AID peptide, equal volumes of equimolar (1-2 μl; 300 μM) protein (SEQ ID NO: 20 and 21, respectively) and AID peptide, having an amino acid sequence of QQLEEDLRGYMSWITQGE (SEQ ID NO: 22) were mixed and equilibrated for 30 min. This solution was screened by hanging drops and gave co-crystals at about the same conditions as described above. Co-crystals have the same space group symmetry as protein crystals with similar unit cell dimensions.
Diffraction data for the SeMet protein crystals and the co-crystals were measured at the ESRF, under standard cryogenic conditions, and processed with HKL software package (Otwinowski, Z., and Minor, W. (1997). Method Enzymol 276, 307-326). A three-wavelength MAD experiment was performed on a single SeMet protein crystal. The anomalous absorption peak, followed by its inflection point and a remote with some anomalous signal were chosen for the wavelengths Scaled data sets for each wavelength were then rescaled by local scaling and 6 selenium sites located using SOLVE (Terwilliger, T. C. (2003). SOLVE and RESOLVE: automated structure solution and density modification. Methods Enzymol 374, 22-37). A clear heavy atom solution was obtained and heavy atom parameters refined to produce experimental phases, using SOLVE. These phases were then used for density modification by RESOLVE (Terwilliger, T. C. (2003). SOLVE and RESOLVE: automated structure solution and density modification. Methods Enzymol 374, 22-37) or SOLOMON (Abrahams, J. P., and Leslie, A. G. W. (1996). Methods used in the structure determination of bovine mitochondrial F-1 ATPase. Acta Cryst D52, 30-42), whose outputs gave two complementary 2.9 Å electron density maps of high quality. A model of β was built with 0 (Jones et al., 1991), and refined with CNS (Br•nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewki, J., Nilges, M., Pannu, N. S., et al (1998). Acta Cryst D54, 905-921). At that point, the model was refined against a non-isomorphous 2.3 Å data set using CNS, REFMAC5 (Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997). Acta Crystallographica Section D—Biological Crystallography 53, 240-255) and ARP (Morris, R. J., Perraakis, A., and Lamzin, V. S. (2003). Methods Enzymol 374, 229-244) with rounds of model rebuilding.
The β/AID peptide co-crystal structure was determined by molecular replacement. Initially, a 3 Å data set measured on the home source was used. Despite the similarity in crystal forms between the β crystals and the co-crystals, rigid body refinement was not sufficient to obtain electron density maps showing the peptide. After molecular replacement (MR) with CNS, using the 2.3 Å refined β model, clear and unmistakable density was found for the bound peptide. MR was required due to a shift of β by several angstroms along one axis. The peptide model was built including changes in β and refined against a 2.2 Å data set collected subsequently. Rounds of model building and refinement were performed as above. The current models have good stereochemistry (Laskowski, R. A., Macarthur, M. W., Moss, D. S., and Thornton, J. M. (1993) Journal of Applied Crystallography 26, 283-291). Both of these crystals utilized protein that bears a mutation in domain I which does not affect function (Opatowsky, Y. Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A. (2003). J Biol Chem 278, 52323-52332).
Diffraction data were collected on an alternative crystal form described in Example 1. This crystal form diffracts to about 3.5 Å and could be obtained using both WT and mutant protein, as well as truncated linker core and linkerless core proteins. Electron density maps and models of the truncated linker core and linkerless tetragonal crystals were obtained by MR with MolRep (Vagin, A., and Teplyakov, A. (2000). Acta Crystallogr D Biol Crystallogr 56 Pt 12, 1622-1624), using the 2.3 Å b model. No gross changes in structure were noted between the WT and mutant protein nor between truncated linker core and linkerless core proteins.
In Vitro Binding Assays
Full length VDCC β2a was mutated by the QuikChange (Stratagene) method. The altered sequence was confirmed by DNA sequencing. Expression, purification, and fluorescence polarization measurements of the mutant proteins were as described (Opatowsky, Y., Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A. (2003). J Biol Chem 278, 52323-52332).
CD Spectroscopy
CD measurements were performed with an Aviv CD spectrometer model 202. Spectra were measured over the range of 260-180 nm at a scan rate of 1 nm/sec. For all measurements, a cell with 1 mm path length was used. The raw data were corrected by subtracting the contribution of the buffer to the CD signal. Data were smoothed and converted to molar ellipticity units. Concentration of peptide was obtained using its predicted extinction coefficient at 280 nm.
Molecular Graphics
FIGS. 4, 6a,b and 7a were prepared with Bobscript (Esnouf, R. M. (1999). Acta Crystallographica Section D-Biological Crystallography 55, 938-940), and Raster3D (Merritt, E. A., and Bacon, D. J. (1997). Macromolecular Crystallography, Pt B 277, 505-524). FIGS. 6c and 7b were prepared with Molscript (Kraulis, P. J. (1991). Journal of Applied Crystallography 24, 946-950) and Raster3D. FIG. 9 was prepared with GRASP (Nicholls, A., Sharp, K. A., and Honig, B. (1991). Proteins 11, 281-296).
Results
The structure of the rabbit β2a functional core was determined by performing a three-wavelength MAD experiment on a single crystal of seleno-methionine substituted protein. Experimental electron density maps at 2.9 Å resolution enabled tracing of the complete molecule with two clearly defined domains, as anticipated. Refinement of the atomic model continued with a 2.3 Å data set. Diffraction data to 3.5 Å was obtained for an alternate crystal form, crystallized with two different forms of the functional core protein. A molecular replacement solution and refinement indicated no significant conformational differences between this and its higher resolution form. Thus, the analysis focused on the high-resolution form.
Subsequently, the β functional core was co-crystallized with an 18 residue AID peptide, whose sequence was: QQLEEDLRGYMSWITQGE (SEQ ID NO: 22), derived from the CaV1.1 AID sequence. This crystal form unequivocally contained the AID peptide, as β also co-crystallized with a fluorescein-labeled peptide that gave bright yellow crystals Diffraction data on the co-crystal was measured to a dmin of 2.2 Å (data presented in Table 3) and the is AID/β complex structure was determined by molecular replacement and rounds of model building and refinement.







TABLE 3







Crystallographic data of the β functional core co-crystallized with AID









Data collection and
SeMet β













phasing statistics
λ1
λ2
λ3
β
AID/β















Wavelength (Å)
0.97905
0.97854
0.90499
0.933
1.0069










Space group
P21212
P21212
P21212


Unit-cell
a = 74.3 
a = 74.1 
a = 72.8 


Parameters
b = 165.7
b = 163.8
b = 168.3


(Å)
c = 34.6 
c = 34.8 
c = 34.2 












Total reflections
84558
76495
63504
71732
89743


Unique reflections
9990
9969
8411
18271
19123


Completeness (%)a
98.8 (94.7)
98.6 (93.7)
95.4 (91.8)
92.8 (87.9)
85.7 (63.2)


Rmerge (%)a,b
 6.1 (21)  
5.9 (19) 
 9.6 (21)  
 4.8 (31)  
 5.9 (31)  


I/σa
30.1 (9.7) 
29.1 (9.7) 
25.6 (10.5)
15.4 (4.3) 
21.3 (3.9) 


Resolution range
50-2.9
50-2.9
50-3.0
50-2.3
50-2.2


(Å)


f′/f′′
−9.68/2.30
−7.68/4.10
−1.33/3.14


Phasing power
0.4
0.6
0.6


(anomalous)


Phasing power
0.5
0.7
0.9


(dispersive)
(λ2 vs λ1)
(λ3 vs λ2)
(λ1 vs λ3)


Figures of Merit

0.39


Beamline
BM-14
BM-14
BM-14
ID-14-2
ID-29


(ESRF)














Refinement statistics
β
AID/β







No. of reflections (working/test)
17294/935 
17364/1283 



dmin (Å)
2.3
2.2



Rwork/Rfree
26.1/28.8
23.3/28.8



Rms deviation from ideality



Bond lengths (Å)
0.013
0.011



Bond angles
1.3°
1.3°



B factors (Å2) (rmsd of bonded
1.1/2.1
1.0/2.1



atoms-main/side chain)



Average B factor (Å2)
62.7
44.1



No. of protein atoms/solvent
2299/91 
2541/130 







aValues for the highest resolution shell are given in parentheses



bRmerge = ΣhkIΣi|Ihkl, i − <I>hkI|/ΣhklΣi|Ihkl, i| where Ihkl is the intensity of a reflection and <I>hkl is the average of all observations of this reflection and its symmetry equivalents






The initial electron density for the bound AID peptide is shown in FIG. 15a. Residues 25-33 (N-terminus), 203-217 (truncated linker), and 416-422 (C-terminus) of β are not visible in the electron density maps.
Example 4
Molecular Architecture of the VDCC β Subunit
The structure revealed a two-domain construction (FIG. 12a), with dimensions 94×54×48 Å, and defined a new variant of the membrane associated guanylate kinase (MAGUK) protein family.
MAGUK proteins have been defined generally by sequences that contain three modules, namely PDZ, SH3 and guanylate kinase-like (Guk) domains. Many of the members contain multiples of the PDZ domain. Family members function as molecular scaffolds, using their various domains to create a web of protein-protein interactions at, or near, the cell membrane. While VDCC β lacked the PDZ domain, its domain I is most similar to MAGUK SH3 domains, and its domain II is a GuK-like domain. β, in fact, represents a minimal MAGUK, having eliminated the PDZ domain and dressed down its GuK domain as detailed below. The structure of the SH3 and GuK domains from a MAGUK protein, PSD-95, served as a critical foil for the structural analysis (McGee, A. W., Dakoji, S. R., Olsen, O., Bredt, D. S., Lim, W. A., and Prehoda, K. E. (2001). Mol Cell 8, 1291-1301; Tavares, G. A., Panepucci, E. H., and Brunger, A. T. (2001). Structural characterization of the intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95. Mol Cell 8, 1313-1325).
The structure of domain I resembles an “adorned” SH3 domain. The fold is shown schematically in FIG. 12b. Long α-helices are appended to the module's amino-terminus and as an insertion between the fourth and fifth strands. Strand E belongs formally to the SH3 architecture while it is nominally part of domain II since limited proteolysis removes the polypeptide connecting helix 2 to strand E, leaving it with the latter domain. Helix 1, not seen in PSD-95, is found in the β structure to partially unwind towards the visible end of its N-terminus. Helix 2 is present in PSD-95 but with a somewhat different orientation. The RMS deviation after superposition between domain I and the Crk SH3 domain (Wu et al., 1995) is 1.3 Å for 52 Ca atoms while the RMSD between domain I and the SH3 of PSD is 1.3 Å for 61 Ca atoms.
A significant difference between domain I, and the SH3 fold, lies in β's unusually long extended RT-loop. Using its length, as seen in FIG. 6a, the β RT-loop takes a different conformation than that of PSD-95, folding in toward the n-Src loop and interacting with W104, thereby occluding the canonical polyproline binding groove. Both helices 1 and 2 make important interactions with parts of the SH3 fold. Helix 1 (E46, A49, L53) contains hydrophobic interactions with the RT loop (residues 80-84) and strands A, B, C and D so that it is well integrated into the fold. At the same time, helix 2 interacts with the hydrophobic core of the domain through interactions from its side chains L125 and M128. Thus, helix 2 projects outward from the SH3 fold precisely where the polyproline lies in its groove on the superposed Crk SH3 domain. Moreover, L125 anchors the helix at about the same position of conserved hydrophobic residues in polyproline ligands i.e. the amino terminus of helix 2 uses similar structural determinants to anchor itself to the domain as do canonical SH3 ligands.
Domain II has strong structural homology to GuK and the GuK-like domains found in MAGUKs (FIG. 14b), and in general, to the P-loop kinase superfamily. The GuK fold has three subdomains: the core, comprised of a parallel five-stranded β-sheet sandwiched by five helices, the mononucleotide binding (NMP) subdomain and the helical lid subdomain. VDCC β contains both the core and lid subdomains but substitutes the NMP subdomain with a structurally unrelated subdomain of about the same length, comprising several short helices, which we have called the “ear” lobe (FIG. 15b). In contrast, the MAGUKs retained the NMP subdomain, comprising a three-stranded sheet and two helices including the ability to bind mononucleotides, in some cases. The RMSD between domain II and yeast Guk (Stehle and Schulz, 1992) is 1.6 Å for 128 Ca atoms and between PSD-95 GuK is 2.2 Å for 86 Ca atoms. These superpositions underline the stronger resemblance of β domain II in the two relevant subdomains to yGuK versus PSD-95.
Since β lacks the NMP subdomain, it is not expected to bind GMP but it remains possible that the ear lobe will bind a small molecule since it does create clefts between itself and the central sheet and itself and strand 5 of domain 1. Another important difference between β and GuK or PSD-95 is the P-loop, also known as the Walker A box, which connects strand 1 and helix 1. As seen in FIG. 13a and FIG. 14b, β's P-loop is significantly longer than PSD and GuK. P loops in the kinase family are important for the binding of ATP phosphate moieties used for phosphotransfer. The composition and conformation of β's loop is pivotal for binding its target as described below. The lid subdomain is retained in all three structures in different orientations. β's lid orientation most closely approximates that of apoGuK (open) versus that of GuK with ADP bound (closed) and PSD-95 (very closed). The difference in orientation may have important consequences for protein target binding. Finally, ATP will not bind A, as β lacks an essential arginine, R131 in yGuK, required for nucleotide binding in all guanylate kinases. In β, the arginine has been replaced with a leucine. This crucial structural difference between GuK and β predicts that phosphotransferase activity has been lost by β and most probably the whole MAGUK family.
How do the two domains interact and how does that organization compare to PSD-95? Strand E of domain I is covalently linked by a short four residue turn to domain II. In addition, other interactions include hydrogen bonds from domain II helix 5 made with the distal loop of domain I. Side chains from the ear lobe and a turn before strand 4 of domain II interact with strand E of domain I. While the individual domains and topology are similar to PSD-95, the actual 3D picture is quite different. Due to PSD-95's very long strand E, the nature of the domain apposition varies. PSD-95's strand F couples with strand E, but is derived from the PSD-95 GuK domain. This is absent in β. The drastically different domain orientation is visualized in FIG. 6c where we have superposed the GuK-like domains. In addition, the domain interface in β is more intimate, burying more accessible surface area (1500 Å2 versus 1200 Å2) than for PSD-95.
Example 5
Protein-Protein Interaction Paradigm for GuKs
Materials and Methods
The GuK domain of the MAGUK family acts as a novel protein-protein interaction domain. Its function is required since several mutant phenotypic alleles of the founding member of the family, Drosophila Discs large (dlg) encode truncated proteins which have their GuK domain deleted. Subsequently, several instances of GuK domains from MAGUK proteins have been shown to bind to various protein targets. Some of the target proteins are motor proteins, used for transporting cargoes to various cellular locations. However, it is unknown how the GuK domains bind these targets.
The AID peptide is found bound to β domain II, consistent with earlier studies that had defined a region of interaction with the AID. The AID forms an α-helix that nestles into a groove on the protein, shaped by the juxtaposition of the lid subdomain and the core subdomain P-loop, helix 1, strand 5 and helix 5. Thus, the AID binds in the same location as does ATP in GuK, visualized in FIG. 14b. Perhaps even more striking, is the superposition of the adenosine moiety from ATP or ADP and the W369 of the AID. While the helical cylinder fits into the groove, the AID has two legs stapling it down. One leg consists of W369 and I370 W369 binds in a deep pit on the domain II surface and makes many interactions with domain II residues (summarized in FIG. 15b). The stereochemistry of W369 is crucial. Most importantly, its Ne1 makes a hydrogen bond to the main chain carbonyl of M246 on β. Consequently, one can easily justify its absolute conservation (FIG. 12b). The second leg consists of G365 and Y366. Y366 is buried completely, making van der Waals interactions with β. Concomitantly, its hydroxyl group makes bifurcated hydrogen-bonds to two water molecules. These, in turn, mediate hydrogen-bonds to β main chain carbonyl (389) or amine (345) groups. These residues are absolutely conserved except in the AID of the electric ray where the glycine is replaced with an arginine. The balance of connections involves van der Waals or hydrophobic interactions. Importantly, interactions between AID and domain II are not localized to one region in sequence space of β so that earlier definitions of the β interacting domain (BID), residues 218 to 250 i.e. strand E of domain I and strand 1, P-loop, and half of helix 1 of domain II are misleading. Three regions in sequence space contribute to the AID binding site, so that for proper binding of the AID one requires almost the complete domain II.
Complexation of the AID with β results in the burial of 1640 Å2 of accessible surface area. The average value for buried surface area amongst protein-protein interaction interfaces is around 1600 Å2. However, the nature of the interface is somewhat unusual. Janin and coworkers, in a survey of protein-protein interaction interfaces, arrived at average number of hydrogen bonds and the amino acid propensity in these interfaces (Lo Conte, L., Chothia, C., and Janin, J. (1999). J Mol Biol 285, 2177-2198). They find that these interfaces are more polar and involve more hydrogen bonds and ion pairs than protein interiors. In this way, the AID/β interface more resembles a protein interior. There are a paucity of hydrogen bonds (four, two being solvent mediated, versus an average of ten), one probable ion pair, and almost all of the remaining interactions are van der Waals or hydrophobic interactions. The β residues in the interface are almost entirely non-polar, while we do not detect a large number of solvent molecules in the interface.
The binding of β to mutagenized AID sequences has been investigated extensively in earlier work. Results of these studies are summarized in FIG. 15c and complement our structural data. They underline the exquisite specificity of W369 and the importance of Y366 and I370. Perturbation of these residues essentially abrogates binding. In general, the importance of residues for binding correlates well their relative burial upon association as computed from the crystal structure. Interestingly, mutation of Gly365 to arginine does not negate binding, suggesting that β will accommodate a bulkier side chain. The sequence conservation of glycine and its stereochemistry may be explained though the dynamics of complexation, described below. Changes in other residues, particularly those of the AID's solvent exposed helical face have little effect on binding.
Mutagenesis of the β subunit and examination of the effects on AID association is less extensive. The BID had several residues mutated (De Waard et al., 1994). Those that had no effect on binding may be rationalized via the structure since they are far from the binding site. Two prolines that were changed to arginines but are distal from the binding site and affected binding should disrupt the protein folding since they are buried. We have characterized two new mutations of β that were designed based on the structure, M246 and L392. M246 is found towards the C-terminal end of the BID, and L392 in the N-terminal end of helix 5. Both residues were changed to alanine and binding constants to AID peptide measured by fluorescence polarization. Both mutants lowered the affinity by one to two orders of magnitude (WT: KD=26±6 nM; M246: 1.7±0.3 mM; L392: 0.34±0.04 mM). These results corroborate our structural data that show the binding site to be composed of sequences outside of the BID.
A comprehensive search of the PDB, using FlexProt (Shatsky et al., 2002), for complexes that showed similarity to the AID/β interface led us to one hit. The crystal structure of the plasmid maintenance system from S. pyogenes constitutes a complex of the z toxin bound to its antitoxin e (Meinhart et al., 2003). While the RMSD is 1.8 Å for 82 out of 183 Ca atoms with a four percent sequence identity, the mode of interaction and even the general architecture of the toxin is quite similar to β's domain II. The toxin has a P-loop kinase fold and generated the proposal that it acts as a phosphotransferase. Mutation of residues putatively required for enzymatic activity abrogated toxicity, suggesting that the catalytic function is responsible for its killing potential. The antitoxin apparently acts by binding the toxin with its first helix inserting into the groove between the toxin's lid and core subdomains. Aromatics bury themselves into positions overlapping, in superpositions, that of W369 and the adenosine base of ATP/ADP of the GuKs.
The comparative structural analysis of the GuK fold lead us to the conclusion that this widely extant protein fold, found in every type of organism, evolved from an enzyme with phosphotransferase capacity to a protein-protein interaction module, losing on its way catalytic activity. Moreover, its ligand-binding site was resculptured to enable protein-protein interactions. Striking examples of this resculpturing are the change of the absolutely required arginine (in yGuK, Arg131) into a leucine in β and the protein target using aromatic side chains in the place of the nucleotide base. In the case of z toxin, catalytic activity is retained and the protein-protein interface facilitates inhibition, while in the case of VDCC β, catalytic activity has been lost to an interface which obtains high affinity and stable binding for its protein target. We propose that the MAGUK family uses the same strategy for binding its protein targets through its GuK domain.
Example 6
VDCC Assembly
The structural determinations herein have important implications for understanding VDCC functional aspects. One aspect, its mechanism for chaperoning the channel has been well-described. It has been shown that the α1 I-II linker contains an endoplasmic retention signal. β then successfully competes with an unknown ER retention protein in binding the linker via the AID, thereby masking the retention signal. This frees the channel to exit the ER and move ultimately to the plasma membrane with β still attached.
Given the crystallographic data that the AID is in an α-helical conformation in the complex, it is important to determine the secondary structure when alone in solution. To address this question, a circular dichroism (CD) spectrum of the peptide was measured. The spectrum, shown in FIG. 16, indicates a random coil structure. This implies that, at least in our in vitro system, the AID peptide undergoes a coil to helix transition during its association with β. We then measured the helical propensity of the AID peptide, by taking a CD spectrum with the peptide in a mixed water/trifluorethanol solution. The spectrum shows significant helical formation. Based on these data, it seems reasonable to posit that the AID associates with β in a coil conformation and undergoes a helical transition upon reaching its high affinity bound state. This hypothesis is consistent with several observations.
One, the nature of the protein-protein interface is largely non-polar and provides the ideal environment for a folding surface, akin to the lower dielectric characteristic of the mixed water/trifluorethanol solvent in the CD measurements. Such an environment is markedly more favorable energetically for the peptide backbone to make its hydrogen-bonds than when faced with an aqueous environment.
Two, the binding kinetics measured for the AID with β (kon ̃2-6·105 M−1·s−1; koff ̃5·10−3 s−1) suggest that the association is not a diffusion-controlled rigid body fast reaction (kon ̃107 M−1·s−1) (Schreiber, G. (2002). Curr Opin Struct Biol 12, 41-47). The values are consistent with a folding step wherein the AID encounters β, low affinity binding occurs and then the AID folds into its helix conformation, locking in the high affinity-binding mode with its myriad interactions. Binding reversibility was established both in vitro and in situ as assessed by electrophysiological assays (Restituito, S., Cens, T., Rousset, M., and Charnet, P. (2001). Biophys J 81, 89-96). In this vein, reversibility could be prevented in an AID mutant replacing the glycine with arginine. Since the data indicates that the AID folds onto β, the conserved glycine provides the reversibility by its low helical propensity.
Three, an established method for detecting AID/β binding uses overlay assays wherein a fusion protein bearing the AID sequence is electrophoresed in a SDS system, transferred to nitrocellulose and labeled β protein is used to probe the filter (Marquart, A. F. V. (1997). FEBS Letters 407, 137-140; Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T. P., and Campbell, K. P. (1994). Nature 368, 67-70). Despite the denaturing manner by which the AID is treated, β succeeds in binding avidly, suggesting that it can associate while the AID is initially unfolded.
Thus, β acts not merely as chaperone, accompanying α1 through the trafficking/processing pathway but also as a chaperonin for the I-II linker section of the channel. Such activity plays a role in ensuring the fidelity of channel assembly. Moreover, helix induction does not occur just in the AID site. Importantly, the protein sequence of the I-II linker, starting from the C-terminal end of membrane domain I i.e. the end of its S6 through the AID sequence motif, is predicted to be one long α-helix by all secondary structure algorithms tested, thus β association with the I-II linker induces helix formation of the AID that is then propagated towards the S6 helix along the polypeptide sequence of the I-II linker.
Example 7
Channel Structure and Function
Several groups have recently made progress towards elucidating the physical organization of VDCCs, using electron microscopy (Murata et al., 2001; Serysheva et al., 2002; Wang et al., 2003; Wolf et al., 2003). A monomeric complex has area dimensions of 110-150×120-165 Å. Identification of the extracellular α2δ has been made, along with a localization of β. The dimensions of the β functional core would fit well into the density. The β subunit, considering its elongated shape, comprises a significant fraction of the intracellular mass of the channel. This observation may be visualized in FIG. 17a, where a schematic α1 subunit has been drawn to an estimated scale with β bound to the α1 I-II linker that contains the AID.
Voltage dependent channels feature two salient molecular characteristics: (i) they are highly ion selective; (ii) they enable permeation of these ions in reaction to the membrane electrical potential. Gating is controlled by the interaction the between voltage sensor (S1-4) subdomain and the helical pore subdomain (S5-6). The conformation of the pore in closed (Kcsa) and open (MthK) positions is known for K+ channels. In order to transition between these two conformational states, it appears that S5 moves, induced by the voltage sensor, which in turn creates lateral torque on S6, causing its helix to bend near a glycine and move out radially, opening the channel. Using this information as a structural template, the S6 of the α1 membrane domain I is followed immediately by the I-II linker. The I-II linker starts with an absolutely conserved sequence (GEF), with the AID positioned exactly 22 residues later in all cases and whose intervening sequences are highly conserved (FIG. 13b). This structure is bound in a noncovalent but stable manner to β. We infer that VDCC gating is not exclusively controlled physically by the voltage-sensor and the pore-lining helices but rather by a complex, conserved and exact physical organization of those elements with the intracellular domains like the I-II linker/β complex. Thus, intracellular elements impinge directly on gating in a manner partially akin to the MthK, KirBac and the Girk channels, gated by ligand binding of intracellular domains.
How might these intracellular elements like the I-II linker/β complex impinge on gating? The AID-β complex structure demonstrates that the AID and β almost seamlessly fuse into a united surface. The resulting electrostatic potential produced shows that β domain II has a patch of negative potential (FIG. 17b), coincident and contiguous to the locale of AID binding. This patch will react to changes in the electrostatic potential of the surrounding environment as a result of membrane depolarization and the channel opening with its flux of Ca2+ ions diffusing from the channel mouth. Hence, the bound β reconfigures the electrostatics of the intracellular side of the channel. Secondly, β orders a part of the channel physically connected to its gate, essentially changing its shape, length and mechanical properties, such as rigidity. Thirdly, through β binding, the gate is now connected to a linker with much greater mass and depending on isoform constrained in its movement by its own independent attachment to the membrane.
Of the known modulatory effects of β, one common denominator is that β seems to shift the equilibrium towards the open channel state i.e. activation. All three aspects of the I-II linker/β just described should facilitate this effect. Upon depolarization, the I-II linker/β negative patch may move in reaction to the changing electrostatic potential of α1, supplementing the radial torque on S6 from the voltage sensor. The presumed helical conformation will lend the requisite rigidity for any movements of the AID-β particle to be communicated to the gate. Also, the additional mass weighing on membrane domain I may destabilize the closed conformation of its S6. Thus, β's action on the channel state is best categorized as allosteric modulation.
Regarding channel inactivation and its kinetics, our structural model fits well with current models that propose a hinged-lid mechanism (Stotz et al., 2004). Here, the AID/β surface serves as the lid, the channel opens, Ca2+ accumulates at the mouth, and attracts the lid by Coulombic forces and in coordination with Ca2+-dependent inactivation determinants of the α1 C-terminus. Subsequently, residues in the AID that are solvent exposed i.e. those not involved in binding β and other residues of the linker find their receptor site possibly in the channel mouth, thereby blocking ion flow and inactivating the channel. According to this inactivation mechanism, the hinge, which enables the lid to swivel may be located at the link between S6 and the I-II linker i.e. the conserved glycine sequence at its start. Another prediction of this model will be the slowing of inactivation if β's movement is constrained through its N-terminus since the “lid” would then not be able to move as easily towards the channel mouth. This notion then explains the slow inactivation of β2α, whose N-terminus is anchored in the membrane by palmitoylation. When the palmitoylation site is mutated, inactivation parameters resemble that of the other isoforms and splice variants.
The structure of β determined represents only part of the molecule. For β3, the functional core represents about 70 percent of the molecule while it is less for the other isoforms. Many studies have demonstrated functions encoded by regions outside of the core, such as inactivation, influenced by the N-terminus or the linker between β domain I and II. Likewise, the C-terminus whose size and sequence varies dramatically between isoforms has been shown to be important for binding other sites on the α1 subunit. The functional core architecture, therefore, maintains the central functions of the molecule. At the same time, extraneous polypeptide regions elaborate functional specificity. Furthermore, functional specificity may be tuned not only by isoform but also by splicing alterations. These splicing permutations fall outside the core architecture.
The molecular architecture of the β functional core indicates that this protein has evolved to maximize protein-protein interactions with other partners. Recent examples of novel proteins that associate with β are the members of the small G-protein subfamily, Gem, Rad, and Rem, and HP1g, involved in gene silencing, indicating its role as nexus of some of the signaling pathways tied to calcium.

## Claims

1. A crystallized voltage-dependent calcium channel (VDCC) β2a subunit functional core, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the VDCC β2a subunit functional core to a resolution of better than 5.0 Angstroms, wherein said crystal is made by vapor diffusion from a VDCC β2a subunit functional core protein concentrated to 12 mg/ml with: a) a reservoir solution of 1.6 M ammonium sulfate, 0.1 M Hepes pH 7.0, 5 mM β-mercaptoethanol and grown at 4° C., or b) a reservoir solution of 3% PEG 20,000, 0.1 M Bicine pH 9, 0.1 M NaCl, 5 mM β-mercaptoethanol and grown at 19° C. and wherein the amino acid sequence of said VDCC β2a subunit functional core has at least 95% identity to SEQ ID NO: 20 or SEQ ID NO: 21, or at least 95% identity to either of said sequences without the four amino acid linker located at residues 114-117 of SEQ ID NO: 20 or 21.

2. The crystal of claim 1, wherein the voltage-dependent calcium channel β2a functional core is a member of the membrane associated guanylate kinase protein family.

3. The crystal of claim 1, wherein the voltage-dependent calcium channel β2a functional core has secondary structural elements that include five beta strands and two helices in the N-terminal domain, designated as strands A, B, C, D, and F, and alpha helices 1 and 2, and five beta strands and eight alpha helices in the C-terminal domain designated as strands 1, 2, 3, 4, and 5, and alpha helices α1, α2, α3, α4.1, α4.2, α5, E1, E2 and E3.

4. A crystallized voltage-dependent calcium channel (VDCC) β2a subunit functional core in complex with an α1 interaction domain (AID) peptide; wherein said AID peptide has at least 92% sequence identity to SEQ ID NO: 22 and wherein the sequence of said VDCC β2a functional core is as set forth in SEQ ID NO: 21; wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of said VDCC β2a functional core:AID complex to a resolution of better than 5.0 Angstroms, and wherein said crystal is made by vapor diffusion from protein stocks of 6-12 mg/mi and a reservoir solution of 1-4% PEG 20K, 0.1 M Bicine pH 9,1-3% 2-methyl-2,4-pentanediol (MPD) and 5 mM β-mercaptoethanol and grown at 19° C.

5. The crystal of claim 4, wherein the voltage-dependent calcium channel β2a functional core is a member of the membrane associated guanylate kinase protein family.

6. The crystal of claim 4, wherein the voltage-dependent calcium channel β2a functional core has secondary structural elements that include five beta strands and two helices in the N-terminal domain, designated as strands A, B, C, D, and F, and alpha helices 1 and 2, and five beta strands and eight alpha helices in the C-terminal domain designated as strands 1, 2, 3, 4, and 5, and alpha helices α1, α2, α3, α4.1, α4.2, α5, E1, E2 and E3.

7. A crystallized voltage-dependent calcium channel (VDCC) β2a subunit functional core in complex with an ccl interaction domain (AID) peptide; wherein the sequence of said AID peptide is as set forth in SEQ ID NO: 22 and wherein said VDCC β2a functional core has at least 95% identity to SEQ ID NO: 21; wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of said VDCC β2a functional core:AID complex to a resolution of better than 5.0 Angstroms, and wherein said crystal is made by vapor diffusion from protein stocks of 6-12 mg/ml and a reservoir solution of 1-4% PEG 20K, 0.1 M Bicine pH 9,1-3% 2-methyl-2,4-pentanediol (MPD) and 5 mM β-mercaptoethanol and grown at 19° C.

8. The crystal of claim 7, wherein the voltage-dependent calcium channel β2a functional core is a member of the membrane associated guanylate kinase protein family.

9. The crystal of claim 7, wherein the voltage-dependent calcium channel β2a functional core has secondary structural elements that include five beta strands and two helices in the N-terminal domain, designated as strands A, B, C, D, and E, and alpha helices 1 and 2, and five beta strands and eight alpha helices in the C-terminal domain designated as strands 1, 2, 3, 4, and 5, and alpha helices α1, α2, α3, α4.1, α4.2, α5, E1, E2 and E3.

10. A crystallized voltage-dependent calcium channel (VDCC) β2a subunit functional core, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the VDCC β2a functional core to a resolution of better than 5.0 Angstroms, said crystal characterized as:
a. Form I of a crystallized VDCC β2a functional core, wherein said crystal has a space group of P41212 with unit cell dimensions of a=b=75.6 Å, c=164.4 Å and α=β=γ=90°; or
b. Form II of a crystallized VDCC β2a functional core, wherein said crystal has a space group of P21212, with unit cell dimensions of a=74.Å, b=163.8Å, c=34.8 Å and α=β=γ=90°;
wherein the amino acid sequence of said VDCC β2a functional core is SEQ ID NO: 21.

11. The crystal of claim 10, wherein the voltage-dependent calcium channel β2a functional core is a member of the membrane associated guanylate kinase protein family.

12. The crystal of claim 10, wherein the voltage-dependent calcium channel β2a functional core has secondary structural elements that include five beta strands and two helices in the N-terminal domain, designated as strands A, B, C, D, and F, and alpha helices 1 and 2, and five beta strands and eight alpha helices in the C-terminal domain designated as strands 1, 2, 3, 4, and 5, and alpha helices α1, α2, α3, α4.1, α4.2, α5, E1, E2 and E3.

13. A crystallized voltage-dependent calcium channel (VDCC) β2a subunit functional core, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the VDCC β2a functional core to a resolution of better than 5.0 Angstroms, wherein said crystal has a space group of P41212 with unit cell dimensions of a=b=76.5 Å, c=164.9 Å and α=β=γ=90°; and the β2a functional core is the amino acid sequence SEQ ID NO: 20 without the four amino acid linker located at residues 114-117.

14. The crystal of claim 13, wherein the voltage-dependent calcium channel β2a functional core is a member of the membrane associated guanylate kinase protein family.

15. The crystal of claim 13, wherein the voltage-dependent calcium channel β2a functional core has secondary structural elements that include five beta strands and two helices in the N-terminal domain, designated as strands A, B, C, D, and F, and alpha helices 1 and 2, and five beta strands and eight alpha helices in the C-terminal domain designated as strands 1, 2, 3, 4, and 5, and alpha helices α1, α2, α3, α4.1, α4.2, α5, E1, E2 and E3.

16. A crystallized voltage-dependent calcium channel (VDCC) β2a subunit functional core in complex with an ccl interaction domain (AID) peptide, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the VDCC β2a functional core:AID peptide complex to a resolution of better than 5.0 Angstroms wherein said crystal has a space group of P21212, with unit cell dimensions of a=72.8 Å, b=168.3 Å, c=34.2 Å and α=β=γ=90°; and wherein the amino acid sequence of said AID peptide is SEQ ID NO: 22 and the β2a functional core is SEQ ID NO: 21.

17. The crystal of claim 16, wherein the voltage-dependent calcium channel β2a functional core is a member of the membrane associated guanylate kinase protein family.

18. The crystal of claim 16, wherein the voltage-dependent calcium channel β2a functional core has secondary structural elements that include five beta strands and two helices in the N-terminal domain, designated as strands A, B, C, D, and F, and alpha helices 1 and 2, and five beta strands and eight alpha helices in the C-terminal domain designated as strands 1, 2, 3, 4, and 5, and alpha helices α1, α2, α3, α4.1, α4.2, α5, E1, E2 and E3.