bluuebunny/crossref_metadata_2025 huggingface dataset:
The astacin family of metalloendopeptidases
Authors: Bond Judith S. , Beynon Robert J.
Published: February 2009

The astacin family of metalloendopeptidases was recognized as a novel family of proteases in the 1990s. The crayfish enzyme astacin was the first characterized and is one of the smallest members of the family. More than 20 members of the family have now been identified. They have been detected in species ranging from hydra to humans, in mature and in developmental systems. Proposed functions of these proteases include activation of growth factors, degradation of polypeptides, and processing of extracellular proteins. Astacin family proteases are synthesized with NH2‐terminal signal and proenzyme sequences, and many (such as meprins, BMP‐1, tolloid) contain multiple domains COOH‐terminal to the protease domain. They are either secreted from cells or are plasma membrane‐associated enzymes. They have some distinguishing features in addition to the signature sequence in the protease domain: HEXXHXXGFXHEXXRXDR. They have a unique type of zinc binding, with pentacoordination, and a protease domain tertiary structure that contains common attributes with serralysins, matrix metalloendopeptidases, and snake venom proteases; they cleave peptide bonds in polypeptides such as insulin B chain and bradykinin and in proteins such as casein and gelatin; and they have arylamidase activity. Meprins are unique proteases in the astacin family, and indeed in the animal kingdom, in their oligomeric structure; they are dimers of disulfide‐linked dimers and are highly glycosylated, type I integral membrane proteins that have many attributes of receptors or integrins with adhesion, epidermal growth factor‐like, and transmembrane domains. The α and β subunits are differentially expressed and processed to yield latent and active proteases as well as membrane‐associated and secreted forms. Meprins represent excellent models of hetero‐ and homo‐oligomeric enzymes that are regulated at the transcriptional and posttranslational levels.
High‐resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer
Authors: Abele U. , Schulz G.E.
Published: February 2009

The structure of adenylate kinase from yeast ligated with the two‐substrate‐mimicking inhibitor Ap5A and Mg2+ has been refined to 1.96 Å resolution. In addition, the refined structure of the same complex with a bound imidazole molecule replacing Mg2+ has been determined at 1.63 Å. These structures indicate that replacing Mg2+ by imidazole disturbs the water structure and thus the complex. A comparison with the G‐proteins shows that Mg2+ is exactly at the same position with respect to the phosphates. However, although the Mg2+ ligand sphere of the G‐proteins is a regular octahedron containing peptide ligands, the reported adenylate kinase has no such ligands and an open octahedron leaving space for the Mg2+ to accompany the transferred phosphoryl group. A superposition of the known crystalline and therefore perturbed phosphoryl transfer geometries in the adenylate kinases demonstrates that all of them are close to the start of the forward reaction with bound ATP and AMP. Averaging all observed perturbed structures gives rise to a close approximation of the transition state, indicating in general how to establish an elusive transition state geometry. The average shows that the in‐line phosphoryl transfer is associative, because there is no space for a dissociative metaphosphate intermediate. As a side result, the secondary dipole interaction in the α‐helices of both protein structures has been quantified.
Mutational analysis of phenylalanine β85 in the valine β6 acceptor pocket during hemoglobin S polymerization
Authors: Adachim Kazuhiko , Reddy Lattupally R. , Surrey Saul , Reddy Konda S.
Published: February 2009

Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at β85 were made and expressed in yeast in an effort to evaluate the role of Phe‐β85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these β385 substitutions do not significantly affect heme‐globin interactions and tetramer helix content. Hb S containing Trp‐β85 had decreased oxygen affinity, whereas those with Leu‐, Ala‐, and Thr‐β85 had increased oxygen affinity. All four supersaturated β85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe‐β88, Ala‐β88, Glu‐β88, or Glu‐β85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477–17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563–31566). Leu substitution at β85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these β85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SFβ85L, Hb SFβ85A, Hb SFβ85T, and Hb sFβ85W are 0.65‐, 2.2‐, 2.5‐ and 3‐fold higher, respectively, than Hb S. These results suggest that the relative order for polymerization of β85 variants (Leu > Phe > Ala > Thr > Trp‐β85) depends on amino acid hydrophobicity rather than stereospecificity of the side chain. These findings are in contrast to previous results for β88 variants. Trp‐β85 in Hb S may affect Val‐β6 acceptor pocket size, but may still accommodate insertion of Val‐β6. These results also strengthen our previous conclusion that β88 amino acid stereospecificity is more critical than that of β85 for insertion of β6 Val.
Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy
Authors: Slupsky Carolyn M. , Smillie Lawrence B. , Sykes Brian D. , Reinach Fernando C.
Published: February 2009

The solution secondary structure of calcium‐saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium‐saturated NTnC (residues 1–90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium‐saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium‐saturated TnC with the X‐ray crystal structure of half‐saturated TnC reveals differences in the ϕ/Ψ angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular ϕ/Ψ angles in the crystal structure, producing a kink in the B helix, whereas in calcium‐saturated TnC, Glu 41 has helical ϕ/Ψ angles, resulting in a straight B helix. The linker between the N and C domains of calcium‐saturated TnC is flexible in the solution structure.
Limited proteolysis of native proteins: The interaction between avidin and proteinase K
Authors: Ellison Derek , Beynon Robert J. , Hinton John , Hubbard Simon J.
Published: February 2009

Avidin is a tetramer of 16‐kDa subunits that have a high affinity for biotin. Proteolysis of native apoavidin by proteinase K results in a limited attack at the loop between β‐strands 3 and 4, involving amino acids 38–43. Specifically, sites of proteolysis are at Thr 40‐Ser 41 and Asn 42‐Glu 43. The limited proteolysis results in an avidin product that remains otherwise intact and which has enhanced binding for 4′‐hydroxyazobenzene‐2‐benzoic acid (HABA), a chromogenic reporter that can occupy the biotin‐binding site. Saturation of the biotin‐binding site with the natural ligand protects avidin from proteolysis, but saturation with HABA enhances the rate of proteolysis of the same site.

Analysis of the three‐dimensional structures of apoavidin and holoavidin reveals that the 3–4 loop is accessible to solvent and scores highly in an algorithm developed to identify sites of proteolytic attack. The structure of holoavidin is almost identical to the apoprotein. In particular, the 3–4 loop has the same structure in the apo and holo forms, yet there are marked differences in proteolytic susceptibility of this region. Evidence suggests that the 3–4 loop is rather mobile and flexible in the apoprotein, and that it becomes constrained upon ligand binding. In one crystal structure of the apoprotein, this loop appears constrained by contacts with symmetry‐related molecules. Structural analyses suggest that the “lid” to the biotin‐binding site, formed by the 3–4 loop, is displaced and made more accessible by HABA binding, thereby enhancing its proteolytic susceptibility.
Solvent accessibility as a predictive tool for the free energy of inhibitor binding to the HIV‐1 protease
Authors: Nauchitel Vladimir , Verde M. Carmen Villa , Sussman Fredy
Published: February 2009

We have developed a simple approach for the evaluation of the free energies of inhibitor binding to the protease of the human immunodeficiency virus (HIV‐1 PR). Our algorithm is based on the observation that most groups that line the binding pockets of this enzyme are hydrophobic in nature. Based on this fact, we have likened the binding of an inhibitor to this enzyme to its transfer from water to a medium of lower polarity. The resulting expression produced values for the free energy of binding of inhibitors to the HIV‐1 PR that are in good agreement with experimental values. The additive nature of this approach has enabled us to partition the free energy of binding into the contributions of single fragments. The resulting analysis clearly indicates the existence of a ranking in the participation of the enzyme's subsites in binding. Although all the enzyme's pockets contribute to binding, the ones that bind the P2‐P'2 span of the inhibitor are in general the most critical for high inhibitor potency. Moreover, our method has allowed us to determine the nature of the functional groups that fit into given enzyme binding pockets. Perusal of the energy contributions of single side chains has shown that a large number of hydrophobic and aromatic groups located in the central portion of the HIV‐1 PR inhibitors present optimal binding. All of these observations are in agreement with experimental evidence, providing a validation for the physical relevancy of our model.
The picornaviral 3C proteinases: Cysteine nucleophiles in serine proteinase folds
Authors: Malcolm Bruce A.
Published: February 2009

The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase‐like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X‐ray crystallography opens the door for the application of the latest advances in computer‐assisted identification and design of anti‐proteinase therapeutic/chemoprophylactic agents.
Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A
Authors: Oblatt‐Montal Myrta , Yamazaki Masahito , Nelson Richard , Montal Mauricio
Published: February 2009

Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel‐forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation‐selective channels when reconstituted in planar lipid bilayers. As predicted, the self‐assembled conductive oligomers express heterogeneous single‐channel conductances. The most frequent openings exhibit single‐channel conductance of 12 and 7 pS in 0.5 M NaC1, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic α‐helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel‐forming activity of intact botulinum toxin A.
C‐terminal specific protein degradation: Activity and substrate specificity of the Tsp protease
Authors: Keiler Kenneth C. , Silber Karen R. , Sauer Robert T. , Downard Kevin M. , Papayannopoulos Ioannis A. , Biemann Klaus
Published: February 2009

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free α‐carboxylate at the C‐terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C‐terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C‐terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.
Kinetics of interaction of partially folded proteins with a hydrophobic dye: Evidence that molten globule character is maximal in early folding intermediates
Authors: Engelhard Markus , Evans Philip A.
Published: February 2009

Interaction with 8‐anilino‐1‐naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates.

Many of these complications can be avoided by experiments where the ANS is introduced only after folding has been allowed to proceed for a variable time. The initial fluorescence intensity after mixing, resulting only from rapid and therefore hopefully relatively nonperturbing interactions with the protein, can be monitored at different refolding times to provide a better reflection of the progress of the reaction, uncomplicated by dye interaction effects. Such studies of the folding of carbonic anhydrase and α‐lactalbumin have been compared with conventional single‐mix experiments and large discrepancies observed. When ANS was present throughout refolding, time‐dependent changes attributed to the formation or reorganization of protein‐ANS complexes were clearly superimposed on those associated with the actual progress of refolding, and the folding kinetics and population of intermediates were also substantially perturbed by the dye. Thus, it is clear that the pulse method, though cumbersome, should be used where refolding reactions are to be probed by dye binding.

The results emphasize that fluorescence enhancement tends to be greatest in early intermediates, in contrast to what, for carbonic anhydrase at least, might appear to be the case from the more conventional experiments. Later intermediates in the folding of both of these proteins actually induce little fluorescence enhancement and therefore may be quite different in nature from equilibrium molten globule states.
Characterization of leucine zipper complexes by electrospray ionization mass spectrometry
Authors: Wendt Hans , Dürr Eberhard , Thomas Richard M. , Przybylski Michael , Bosshard Hans Rudolf
Published: February 2009

The development of “soft” ionization methods has enabled the mass spectrometric analysis of higher‐order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI‐MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI‐MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled‐coil structures. ESI‐MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4‐pl of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac‐EYEALEKKLAAX1EAKX2QALEKKLEALEHG‐amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI‐MS as could the dimeric nature of GCN4‐pl. The formation of heteromeric two‐ and three‐stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4‐pl and LZ, was demonstrated by ESI‐MS and confirmed by fluorescence quenching experiments on fluorescein‐labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled‐coil domains.
Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy
Authors: Pountney Dean L. , Henehan Colin J. , Vasak Milan
Published: February 2009

Far‐UV CD, 1H‐NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well‐characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 μg total protein) and, unlike either CD or 1H‐NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.
The distribution of α‐helix propensity along the polypeptide chain is not conserved in proteins from the same family
Authors: Muñoz Victor , Blanco Francisco J. , Serrano Luis
Published: February 2009

We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of α‐helices. We have analyzed by CD the conformation of peptides corresponding to the five α‐helices of two α/β parallel proteins (ComA and Ara). The large α‐helical population of peptide ComA‐4 detected by CD in aqueous solution has been confirmed by NMR. These proteins are members of the CheY and P21‐ras families, respectively, which have been studied previously in the same way (Muñoz V, Jiménez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275–296). Comparison of the helical content of equivalent peptides reveals that protein α‐helix propensity profiles are not conserved. Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY). However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.
Predicting oligomerization states of coiled coils
Authors: Woolfson Derek N. , Alber Tom
Published: February 2009

An algorithm based on the profile method was developed that faithfully distinguishes between the amino acid sequences of dimeric and trimeric coiled coils. Normalized sequence profiles derived from nonhomologous, two‐and three‐stranded, coiled‐coil sequences with unambiguous registers were used to assign dimer and trimer propensities to test sequences. The difference between the dimer and trimer profile scores accurately reflected the preferred oligomerization state. The method relied on two strategies that may be generally applicable to profile calculations —profile values of solvent‐exposed residues and of amino acids that were underrepresented in the database were given zero weight. Differences between the dimer and trimer profiles revealed sequence patterns that match and extend experimental studies of oligomer specification.
Purification and preliminary X‐ray crystallographic studies of recombinant L‐ribulose‐5‐phosphate 4‐epimerase from escherichia coli
Authors: Andersson Arnold , Schneider Gunter , Lindqvist Ylva
Published: February 2009

The araD gene from Escherichia coli, coding for L‐ribulose‐5‐phosphate 4‐epimerase, was overexpressed and the resulting enzyme was purified to homogeneity. Crystals of L‐ribuIose‐5‐phosphate 4‐epimerase, obtained with 4.0 M sodium formate as precipitant, belong to space group P4212 with unit cell dimensions a = b = 107.8 Å and c = 281.4 Å and diffract to at least 2.2 Å resolution. Density measurements of these crystals are consistent with eight subunits in the asymmetric unit.
Crystallization and preliminary X‐ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor
Authors: Keefe Lisa J. , Ginell Stephan L. , Westbrook Edwin M. , Anderson Carl W.
Published: February 2009

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine‐substituted) has been crystallized in the presence of the serotype 12, 11‐residue peptide cofactor. The crystals (space group P3121 or P3221, one molecule per asymmetric unit, a = b = 41.3 Å, c = 197.0 Å) grew in solutions containing 20–40% 2‐methyl‐2,4‐pentanediol (MPD), 0.1–0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0–7.5. Diffraction data (84% complete to 2.2 Å resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 × 1,024 pixel array) charge‐coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine‐substituted proteinase, 65% complete to 2.0 Å resolution with Rmerge values ranging 0.05–0.07, have been collected at three X‐ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.
Structural similarity between the pleckstrin homology domain and verotoxin: The problem of measuring and evaluating structural similarity
Authors: Orengo C.A. , Swindells M.B. , Michie A.D. , Zvelebil M.J. , Driscoll P.C. , Waterfield M.D. , Thornton J.M.
Published: February 2009

An unexpected structural similarity is described between the pleckstrin homology (PH) domain and verotoxin. This similarity has escaped detection primarily due to the differences in topology that exist between the two proteins. By comparing this result with two previously reported similarities for the PH domain, one with the lipocalins and another with the FK506 binding protein, we discuss the problems of measuring and assessing structural similarities.
Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain
Authors: Helms Larry R. , Wetzel Ronald
Published: February 2009

It is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.
Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues
Authors: Colette Daubner S. , Ehrlich Joel I. , Ramsey Andrew J. , Fitzpatrick Paul F.
Published: February 2009

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild‐type enzyme, about 0.6 atoms/sub‐unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild‐type enzyme, and slightly altered V/Km values for both tyrosine and 6‐methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild‐type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.
Modeling conformational changes in cyclosporin A
Authors: O'Donohue Mark F. , Burgess Antony W. , Treutlein Herbert R , Walkinshaw Malcolm D.
Published: February 2009

NMR and X‐ray structures for the immunosuppressant cyclosporin A (CsA) reveal a remarkable difference between the unbound (free) conformation in organic solvents and the conformation bound to cyclophilin. We have performed computer simulations of the molecular dynamics of CsA under a variety of conditions and confirmed the stability of these two conformations at room temperature in water and in vacuum. However, when the free conformation was modeled in vacuum at 600 K, a transition pathway leading to the bound conformation was observed. This involved a change in the cis MeLeu‐9 peptide bond to a trans conformation and the movement of the side chains forming the dominant hydrophobic cluster (residues MeBmt‐1, MeLeu‐4, MeLeu‐6, and MeLeu‐10) to the opposite side of the plane formed by the backbone atoms in the molecular ring. The final conformation had a backbone RMS deviation from the bound conformation of 0.53 Å and was as stable in dynamics simulations as the bound conformation. Our calculations allowed us to make a detailed analysis of a transition pathway between the free and the bound conformations of CsA and to identify two distinct regions of coordinated movement in CsA, both of which underwent transitions independently.
Molecular modeling of the GM‐CSF and IL‐3 receptor complexes
Authors: Lyne P.D. , Bamborough P. , Duncan D. , Richards W.G.
Published: February 2009

A model for the structure of the cytokine interleukin‐3 (IL‐3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin‐5 and granulocyte macrophage colony stimulating factor (GM‐CSF). In addition, models of the receptor complexes of GM‐CSF and IL‐3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM‐CSF and IL‐3 to their receptors.
Urease activity in the crystalline state
Authors: Moncrief Mary Beth C. , Hausinger Robert P. , Hom Louis G. , Jabri Evelyn , Andrew Karplus P.
Published: February 2009

Crystalline Klebsiella aerogenes urease was found to have less than 0.05% of the activity observed for the soluble enzyme under standard assay conditions. Li2SO4, present in the crystal storage buffer at 2 M concentration, was shown to inhibit soluble urease by a mixed inhibition mechanism (Ki's of 0.38 ± 0.05 M for the free enzyme and 0.13 ± 0.02 M for the enzyme‐urea complex). However, the activity of crystals was less than 0.5% of the expected value, suggesting that salt inhibition does not account for the near absence of crystalline activity. Dissolution of crystals resulted in ˜43% recovery of the soluble enzyme activity, demonstrating that protein denaturation during crystal growth does not cause the dramatic diminishment in the catalytic rate. Finally, crushed crystals exhibited only a threefold increase in activity over that of intact crystals, indicating that the rate of substrate diffusion into the crystals does not significantly limit the enzyme activity. We conclude that urease is effectively inactive in this crystal form, possibly due to conformational restrictions associated with a lid covering the active site, and propose that the small amounts of activity observed arise from limited enzyme activity at the crystal surfaces or trace levels of enzyme dissolution into the crystal storage buffer.
Role of the N‐terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin
Authors: Findlay Wendy A. , Gradwell Michael J. , Bayley Peter M.
Published: February 2009

The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk‐MLCK) target sequence has been studied using 1H‐NMR. The 18‐residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C‐domain and the Phe 17 side chain with the N‐domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H‐NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C‐domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C‐domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H‐NMR spectra of CaM bound to WFF, a Trp 4 → Phe analogue (FFF), or a Trp 4 → Phe/Phe 17 → Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C‐domain and the side chain in position 17 interacting with the N‐domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34: 2087–2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N‐domain of CaM.
Addition of side chain interactions to modified Lifson‐Roig helix‐coil theory: Application to energetics of Phenylalanine‐Methionine interactions
Authors: Stapley Benjamin J. , Rohl Carol A. , Doig Andrew J.
Published: February 2009

We introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson‐Roig helix‐coil theory of Doig et al. (1994, Biochemistry 33:3396–3403). The helix/coil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields ΔG for the Phe‐Met orientation to be −0.75 kcal·mol−1, whereas that for the Met‐Phe orientation is −0.54 kcal·mol−1. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe‐Met i, i + 4 at the N‐terminus, the C‐terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain‐side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix.
Crystallization and preliminary X‐ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides
Authors: Roberts David L. , Runquist Jennifer A. , Miziorko Henry M. , Kim Jung‐Ja P.
Published: February 2009

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 Å. Based on the assumption of one 32‐kDa monomer per asymmetric unit, the Vm value is 2.83 Å3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 Å resolution.
Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor
Authors: Van Doren Steven R. , Kurochkin Alexander V. , Hu Weidong , Ye Qi‐Zhuang , Johnson Linda L. , Hupe Donald J. , Zuiderweg ERIK R.P.
Published: February 2009

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium‐resolution structure of a 20‐kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed β‐sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine‐containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166‐residue protein fragment complexed with a 4‐residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 Å. The structure has good stereochemical properties, including its backbone torsion angles. The β‐sheet and α‐helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X‐ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 55:8207‐8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 Å when three loop segments are disregarded. The hydroxamate‐substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S1' subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S2' surface subsite, and a third hydrophobic side chain (P3') lies at the surface.
Crystallization and preliminary X‐ray analysis of L‐2‐haloacid dehalogenase from xanthobacter autotrophicus GJ10
Authors: Ridder Ivo S. , Rozeboom Henriëtte J. , Dijkstra Bauke W. , Kingma Jaap , Janssen Dick B.
Published: February 2009

Haloacid dehalogenases are enzymes that cleave carbon‐chlorine or carbon‐bromine bonds of 2‐haloalkanoates. X‐ray‐quality crystals of L‐2‐haloacid dehalogenase from the 1, 2‐dichloroethane‐degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8‐7.0, using macroseeding techniques. The crystals, which diffract in the X‐ray beam up to 2.0 Å resolution, belong to the spacegroup C2221. Cell parameters are a = 58.8 Å, b = 93.1 Å, c = 84.2 Å. A native data set to 2.3 Å has been collected, with a completeness of 97% and an Rsym of 6.0%.
Ethanol utilization regulatory protein: Profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion
Authors: Nicholas Hugh B. , Persson Bengt , Jörnvall Hans , Hempel John
Published: February 2009

The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 746:145‐158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion.
The lipocalin XLCpl1 expressed in the neural plate ofXenopus laevisembryos is a secreted retinaldehyde binding protein
Authors: Lepperdinger Günter , Strobl Birgit , Weber Anita , Mollay Christa , Jilek Alexander , Thalhamer Josef , Flöckner Hannes
Published: February 2009

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate ofXenopus laevisembryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpll, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. TheXlcpl1cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpll‐expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor‐controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.
Free energy mapping of class I MHC molecules and structural determination of bound peptides
Authors: Sezerman Ugur , Vajda Sandor , Delisi Charles
Published: February 2009

Free energy maps of the binding site are constructed for class I major histocompatibility complex (MHC) proteins, by rotating and translating amino acid probes along the cleft, and performing a side‐chain conformational search at each position. The free energy maps are used to determine favorable residue positions that are then combined to form docked peptide conformations. Because the generic backbone structural motif of peptides bound to class I MHC is known, the mapping is restricted to appropriate regions of the site, but allows for the sometimes substantial variations in backbone and side‐chain conformations. In a test demonstrating the quality of predictions for a known MHC site using only a rotational and conformational search, we started from the crystal structure of the HIV‐1 gp120/HLA‐A2 complex, and predicted the HLA‐A2 bound structures of peptides from the influenza matrix protein, the HIV‐1 reverse transcriptase, and the human T cell leukemia virus. The calculated peptides are at 1.6, 1.3, and 1.4 Å all‐atom RMSDs from their respective crystal structures (Madden DR, Garboczi DN, Wiley DC, 1993). A further test, which also included a local translational search, predicted structures across MHCs. In particular, we obtained the Kb/SEV‐9 complex (Fremont DH et al., 1992, Science 257:919‐927) starting with the complex between HLA‐B27 and a generic peptide (Madden DR, Gorga JC, Strominger JL, Wiley DC, 1991, Nature (Lond) 555:321‐325), with an all‐atom RMSD of 1.2 Å, indicating that the docking procedure is essentially as effective for predictions across MHCs as it is for determinations within the same MHC, although at substantially greater computational cost. The requirements for further improvement in accuracy are identified and discussed briefly.
In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: Implications for protein folding and assembly
Authors: Zhang Pinghui , Schachman H.K.
Published: February 2009

Because the N‐ and C‐terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild‐type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild‐type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16‐21 μmol carbamoylaspartate per μg per h, compared with 15 for wild‐type ATCase, and the values for K0.5 for the variants were 4‐17 mM aspartate, whereas wild‐type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild‐type enzyme. As observed with wild‐type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild‐type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N‐ and C‐termini exhibited the homotropic and heterotopic effects characteristic of wild‐type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N‐(phosphonacetyl)‐L‐aspartate (PALA) was also virtually identical to that obtained with wild‐type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand‐promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N‐ and C‐termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.
A recipe for designing water‐soluble, β‐sheet‐forming peptides
Authors: Mayo Kevin H. , Ilyina Elena , Park Henry
Published: February 2009

Based on observations of solubility and folding properties of peptide 33‐mers derived from the β‐sheet domains of platelet factor‐4 (PF4), interleukin‐8 (IL‐8), and growth related protein (Gro‐α), as well as other β‐sheet‐forming peptides, general guidelines have been developed to aid in the design of water soluble, self‐association‐induced β‐sheet‐forming peptides. CD, 1H‐NMR, and pulsed field gradient NMR self‐diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native‐like β‐sheet tetramers and is sparingly soluble above pH 6. IL‐8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, nativelike β‐sheet dimers at higher pH. Gro‐α peptide is soluble at all pH values, yet displays no discernable β‐sheet structure even when diffusion data indicate dimer‐tetramer aggregation. A recipe used in the de novo design of water‐soluble β‐sheet‐forming peptides calls for the peptide to contain 40‐50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noneharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33‐residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form β‐sheet structures to varying degrees, and self‐associate. One peptide folds as a stable, compact β‐sheet tetramer, whereas the others are transient β‐sheet‐containing aggregates.