bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR study of Ni2+ binding to the H‐N‐H endonuclease domain of colicin E9
Authors: Hannan Jonathan P. , Whittaker Sara B.‐M. , Davy Sharon L. , Kühlmann Ulrike C. , Pommer Ansgar J. , Hemmings Andrew M. , James Richard , Kleanthous Colin , Moore Geoffrey R.
Published: February 2009

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2− at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)n]2−. Dialysis against ≥0.2 MNaCl was required to remove the [Ni(EDTA)(H2O)n]2−. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni (EDTA)(H2O)n]2− should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X‐ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.
Stable proline box motif at the N‐terminal end of α‐helices
Authors: Viguera A.R. , Serrano L.
Published: February 2009

We describe a novel N‐terminal α‐helix local motif that involves three hydrophobic residues and a Pro residue (Pro‐box motif). Database analysis shows that when Pro is the N‐cap of an α‐helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an α‐helix, but not when Pro is at position N1. N‐cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N‐cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N‐cap and helix formers results in a small overall stabilization of the α‐helix with respect to other peptides having Gly as the N‐cap and Ala at N3 and N4. This suggests that the Pro‐box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position.
Monomer‐trimer equilibrium of the ectodomain of SIV gp41: Insight into the mechanism of peptide inhibition of HIV infection
Authors: Caffrey Michael , Kaufman Joshua , Stahl Stephen , Wingfield Paul , Gronenborn Angela M. , Clore G. Marius
Published: February 2009

The monomer‐trimer equilibrium of the ectodomain of SIV gp41 (residues 27–149, e‐gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e‐gp41 trimer is 3.1 × 1011 M−2. The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C‐labeled e‐gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e‐gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer‐trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.
The acid‐induced folded state of Sac7d is the native state
Authors: Bedell Jennifer L. , Mccrary Bradford S. , Edmondson Stephen P. , Shriver John W.
Published: February 2009

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J, Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203–224). Both near‐ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid‐ and salt‐induced folded states have a native fold and are not molten globular. 1H, 15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid‐, and salt‐induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through‐bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid‐ and salt‐induced folded states are identical to those observed in the native. The radius of gyration of the acid‐induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X‐ray scattering. We conclude that acid‐induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid‐induced folding except that the A‐state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.
A 16‐amino acid peptide from human α2‐macroglobulin binds transforming growth factor‐β and platelet‐derived growth factor‐BB
Authors: Webb Donna J. , Roadcap David W. , Dhakephalkar Anita , Gonias Steven L.
Published: February 2009

α2‐Macroglobulin (α2M) is a major carrier of transforming growth factor‐β (TGF‐β) in vitro and in vivo. By screening glutathione S‐transferase (GST) fusion proteins with overlapping sequences, we localized the TGF‐β‐binding site to aa 700–738 of the mature human α2M subunit. In separate experiments, we screened overlapping synthetic peptides corresponding to aa 696–777 of α2M and identified a single 16‐mer (718–733) that binds TGF‐β1. Platelet‐derived growth factor‐BB (PDGF‐BB) bound to the same peptide, even though TGF‐β and PDGF‐BB share almost no sequence identity. The sequence of the growth factor‐binding peptide, WDLVVVNSAGVAEVGV, included a high proportion of hydrophobic amino acids. The analogous peptide from murinoglobulin, a human α2M homologue that does not bind growth factors, contained only three nonconservative amino acid substitutions; however, the MUG peptide failed to bind TGF‐β1 and PDGF‐BB. These results demonstrate that a distinct and highly‐restricted site in α2M, positioned near the C‐terminal flank of the bait region, mediates growth factor binding. At least part of the growth factor‐binding site is encoded by exon 18 of the α2M gene, which is notable for a 5′ splice site polymorphism that has been implicated in Alzheimer's Disease.
For the record: Chemical modification of a variant of human MIP‐1α; implications for dimer structure
Authors: Ashfield J. T. , Fisher J. , Meyers T. , Lowne D. , Czaplewski L. G. , Dudgeon T. , Varley P. G. , Arnold J. R. P. , Tan P. , Yang J.‐C.
Published: February 2009

A sequence variant of human MIP‐1α, in which Asp26 has been replaced by Ala, has been chemically modified by the addition of 13C‐labeled methyl groups at each of the lysine residues and the N‐terminus. The sites of methylation have been verified by a combination of MALDI‐TOF mass spectrometric experiments and tryptic digestion followed by N‐terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra‐centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP‐1α D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP‐3, rather than the elongated or cylindrical structure determined for dimers of huMIP‐1β and RANTES.
Structural characterization of a mutant peptide derived from ubiquitin: Implications for protein folding
Authors: Zerella Rosa , Williams Dudley H. , Chen Pei‐Yeh , Evans Philip A. , Raine Andrew
Published: February 2009

The formation of the N‐terminal β‐hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1–17 of ubiquitin folds autonomously and is likely to have a native‐like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1–17)T9D, the native conformation is stabilized with respect to the wild‐type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U(1–17)T9D peptide does indeed form a hairpin with a native‐like register and a type I turn with a G1 β‐bulge, as in the full‐length protein. The reason for the greater stability of the U(1–17)T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys11, which may indicate that a charge‐charge interaction between these residues is responsible.
Position effect of cross‐strand side‐chain interactions on β‐hairpin formation
Authors: Santiveri Clara M. , Rico Manuel , Jiménez M. Angeles
Published: February 2009

Previous conformational analysis of 10‐residue linear peptides enabled us to identify some cross‐strand side‐chain interactions that stabilize β‐hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N‐ and C‐termini of these 10‐residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on β‐hairpin formation, we have designed two 15‐residue β‐hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15‐residue peptides suggest that the influence of stabilizing factors for β‐hairpin formation, in particular, cross‐strand side‐chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in β‐hairpin folding.
A post‐translational modification of nuclear proteins, NG,NG‐dimethyl‐Arg, found in a natural HLA class I peptide ligand
Authors: Yagüe Jesús , Vázquez Jesús , Castro JOSÉ A. LÓPez De
Published: February 2009

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self‐tolerance and induction of cytotoxic T‐cell responses against intracellular parasites. Despite frequent and diverse post‐translational modification of eukaryotic cell proteins, very few class I‐bound peptides with post‐translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA‐B39 (B*3910) derived from an RNA‐binding nucleoprotein that carried NG,NG‐dimethyl‐Arg. Although common among RNA‐binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl‐Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post‐translationally modified ligand was a prominent component (1–2%) of the B*3910‐bound peptide repertoire. The dimethyl‐Arg residue was located in a central position of the peptide, amenable to interacting with T‐cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post‐translationally modified residue may have a major influence on the antigenic properties of this natural ligand.
Automated search of natively folded protein fragments for high‐throughput structure determination in structural genomics
Authors: Kuroda Yutaka , Tani Kazutoshi , Matsuo Yo , Yokoyama Shigeyuki
Published: February 2009

Structural genomic projects envision almost routine protein structure determinations, which are currently imaginable only for small proteins with molecular weights below 25,000 Da. For larger proteins, structural insight can be obtained by breaking them into small segments of amino acid sequences that can fold into native structures, even when isolated from the rest of the protein. Such segments are autonomously folding units (AFU) and have sizes suitable for fast structural analyses. Here, we propose to expand an intuitive procedure often employed for identifying biologically important domains to an automatic method for detecting putative folded protein fragments. The procedure is based on the recognition that large proteins can be regarded as a combination of independent domains conserved among diverse organisms. We thus have developed a program that reorganizes the output of BLAST searches and detects regions with a large number of similar sequences. To automate the detection process, it is reduced to a simple geometrical problem of recognizing rectangular shaped elevations in a graph that plots the number of similar sequences at each residue of a query sequence. We used our program to quantitatively corroborate the premise that segments with conserved sequences correspond to domains that fold into native structures. We applied our program to a test data set composed of 99 amino acid sequences containing 150 segments with structures listed in the Protein Data Bank, and thus known to fold into native structures. Overall, the fragments identified by our program have an almost 50% probability of forming a native structure, and comparable results are observed with sequences containing domain linkers classified in SCOP. Furthermore, we verified that our program identifies AFU in libraries from various organisms, and we found a significant number of AFU candidates for structural analysis, covering an estimated 5 to 20% of the genomic databases. Altogether, these results argue that methods based on sequence similarity can be useful for dissecting large proteins into small autonomously folding domains, and such methods may provide an efficient support to structural genomics projects.
Structural comparison of Ntn‐hydrolases
Authors: Oinonen Carita , Rouvinen Juha
Published: February 2009

The Ntn‐hydrolases (N‐terminal nucleophile) are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N‐terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is almost completely absent, the enzymes share a similar αββα‐core structure. The central β‐sheets in the core were found to have different packing angles, ranging from 5 to 35°. In the Ntn‐hydrolases under study, eight totally conserved secondary structure units were found (region C). Five of them were observed to contain the greatest number of conserved and functionally important residues and are therefore crucial for the structure and function of Ntn‐hydrolases. Two additional regions, consisting of secondary structure units (regions A and B), were found to be in structurally similar locations, but in different orders in the polypeptide chain. The catalytic machinery is located in the structures in a similar manner, and thus the catalytic mechanisms of all of the enzymes are probably similar. However, the substrate binding and the oxyanion hole differed partially.
Presence of closely spaced protein thiols on the surface of mammalian cells
Authors: Donoghue Neil , Yam Patricia T.W. , Jiang Xing‐Mai , Hogg Philip J.
Published: February 2009

It has been proposed that certain cell‐surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane‐impermeable trivalent arsenical to identify closely spaced thiols in cellsurface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4‐(N‐(S‐glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell‐surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the γ‐glutamyl residue of GSAO (GSAO‐B). Incorporation of GSAO‐B into proteins was assessed by measuring the biotin using streptavidin‐peroxidase. Up to 12 distinct proteins were labeled with GSAO‐B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO‐B. These findings demonstrate that the cell‐surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active.
Enhancement by Mg2+ of domain specificity in Ca2+‐dependent interactions of calmodulin with target sequences
Authors: Martin Stephen R. , Masino Laura , Bayley Peter M.
Published: February 2009

Mg2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca2+‐dependent target interactions. The binding of Mg2+ does, however, produce significant spectroscopic changes in residues located in the Ca2+‐binding loops, and the Mg‐calmodulin complex is significantly different from apo‐calmodulin in loop conformation. Direct measurement of Mg2+ binding constants, and the effects of Mg2+ on Ca2+ binding to calmodulin, are consistent with specific binding of Mg2+, in competition with Ca2+. Mg2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg2+ has a direct role in conferring stability on both domains of apo‐calmodulin. Apo‐calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with Kd ∼ 70‐90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg2+, in marked contrast to the strong enhancement of peptide affinity induced by Ca2+. Under conditions of stimulation and increased [Ca2+], Mg2+ has a role in directing the mode of initial target binding preferentially to the C‐domain of calmodulin, due to the opposite relative affinities for binding of Ca2+ and Mg2+ to the two domains. Mg2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca2+ threshold for enzyme activation and increases the importance of a partially Ca2+‐saturated calmodulin‐target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca2+‐dependent target interactions.
Rationale for Bcl‐XL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies
Authors: Petros Andrew M. , Nettesheim David G. , Wang Yi , Olejniczak Edward T. , Meadows Robert P. , Mack Jamey , Swift Kerry , Matayoshi Edmund D. , Zhang Haichao , Fesik Stephen W. , Thompson Craig B.
Published: February 2009

The three‐dimensional structure of the anti‐apoptotic protein Bcl‐XL complexed to a 25‐residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl‐XL bound to a 16‐residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl‐XL. However, based upon site‐directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25‐mer for Bcl‐XL compared to the Bad 16‐mer. Rather, the increased helix propensity of the Bad 25‐mer is primarily responsible for its greater affinity for Bcl‐XL. Based on this observation, a pair of 16‐residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl‐XL. Both peptides showed an increase in helix propensity compared to the wild‐type and exhibited an enhanced affinity for Bcl‐XL.
The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding
Authors: Teplova Marianna , Tereshko Valentina , Egli Martin , Sanishvili Ruslan , Joachimiak Andrzej , Bushueva Tatyana , Anderson Wayne F.
Published: February 2009

The yrdC family of genes codes for proteins that occur both independently and as a domain in proteins that have been implicated in regulation. An example for the latter case is the sua5 gene from yeast. Sua5 was identified as a suppressor of a translation initiation defect in cytochrome c and is required for normal growth in yeast (Na JG, Pinto I, Hampsey M, 1992, Genetics 11:791‐801). However, the function of the Sua5 protein remains unknown; Sua5 could act either at the transcriptional or the posttranscriptional levels to compensate for an aberrant translation start codon in the cyc gene. To potentially learn more about the function of YrdC and proteins featuring this domain, the crystal structure of the YrdC protein from Escherichia coli was determined at a resolution of 2.0 Å. YrdC adopts a new fold with no obvious similarity to those of other proteins with known three‐dimensional (3D) structure. The protein features a large concave surface on one side that exhibits a positive electrostatic potential. The dimensions of this depression, its curvature, and the fact that conserved basic amino acids are located at its floor suggest that YrdC may be a nucleic acid binding protein. An investigation of YrdC's binding affinities for single‐ and double‐stranded RNA and DNA fragments as well as tRNAs demonstrates that YrdC binds preferentially to double‐stranded RNA. Our work provides evidence that 3D structures of functionally uncharacterized gene products with unique sequences can yield novel folds and functional insights.
The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus
Authors: Thompson Gary S. , Radford Sheena E. , Leung Yun‐Chung , Redfield Christina , Ferguson Stuart J.
Published: February 2009

The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35 ± 0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double‐wound Greek‐key fold with two α‐helices toward its C‐terminus, similar to that of its oxidized counterpart determined by X‐ray crystallography. Comparison of the Cu(I) solution structure with the X‐ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.
Peptide and metal ion‐dependent association of isolated helix‐loop‐helix calcium binding domains: Studies of thrombic fragments of calmodulin
Authors: Brokx Richard D. , Vogel Hans J.
Published: February 2009

Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium‐binding regulatory protein, has been cleaved by thrombin to create two fragments, TM1 (1‐106) and TM2 (107‐148). NMR and CD results indicate that TM1 and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix‐loop‐helix domains, but rather within an α‐helix. Cadmium‐113 NMR results show that this complex has enhanced metal‐ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM‐binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl‐13C‐Met‐labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium‐113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM‐dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix‐loop‐helix Ca2+‐binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.
NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor‐1 (AMCI‐1): Structural similarity with Ascaris protease inhibitors
Authors: Cierpicki Tomasz , Bania Jacek , Otlewski Jacek
Published: February 2009

The three‐dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI‐1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular β‐sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI‐1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH‐induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI‐1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu → Gln substitution at the P1′ position in AMCI‐1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI‐1 structure.
Change in backbone torsion angle distribution on protein folding
Authors: Petrescu Andrei‐José , Calmettes Patrick , Durand Dominique , Receveur Veronique , Smith Jeremy C.
Published: February 2009

Understanding protein folding requires the determination of the configurational space accessible to the protein at different stages in folding. Here, computer simulation analysis of small angle neutron scattering results is used to probe the change in the distribution of configurations on strong denaturation of a globular protein, phosphoglycerate kinase, in 4 M guanidine hydrochloride solution. To do this atomic‐detail ensembles of the unfolded protein chain are modeled and their scattering profiles compared with the experiment. The local conformational statistics are found to strongly influence the experimental intensity at scattering vectors between 0.05 and 0.3 Å−1. Denaturation leads to a reduction in the protein atom‐pair distance distribution function over the ∼3‐15 Å region that is associated with a quantifiable shift in the backbone torsional angle (φ,ψ) distribution toward the β region of the Ramachandran plot.
Structural requirements of pyrroloquinoline quinone dependent enzymatic reactions
Authors: Oubrie Arthur , Dijkstra Bauke W.
Published: February 2009

On the basis of crystal structures of the pyrroloquinoline quinone (PQQ) dependent enzymes methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (s‐GDH), different catalytic mechanisms have been proposed. However, several lines of biochemical and kinetic evidence are strikingly similar for both enzymes. To resolve this discrepancy, we have compared the structures of these enzymes in complex with their natural substrates in an attempt to bring them in line with a single reaction mechanism. In both proteins, PQQ is located in the center of the molecule near the axis of pseudo‐symmetry. In spite of the absence of significant sequence homology, the overall binding of PQQ in the respective active sites is similar. Hydrogen bonding interactions are made with polar protein side chains in the plane of the cofactor, whereas hydrophobic stacking interactions are important below and above PQQ. One Arg side chain and one calcium ion are ligated to the ortho‐quinone group of PQQ in an identical fashion in either active site, in agreement with their proposed catalytic function of polarizing the PQQ C5‐O5 bond. The substrates are bound in a similar position above PQQ and within hydrogen bond distance of the putative general bases Asp297 (MDH) and His144 (s‐GDH). On the basis of these similarities, we propose that MDH and s‐GDH react with their substrates through an identical mechanism, comprising general base‐catalyzed hydride transfer from the substrate to PQQ and subsequent tautomerization of the PQQ intermediate to reduced PQQ.
Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three‐dimensional structure
Authors: Tomschy Andrea , Tessier Michel , Wyss Markus , Brugger Roland , Broger Clemens , Schnoebelen Line , Van Loon Adolphus P.G.M. , Pasamontes Luis
Published: February 2009

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49‐68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U[mg protein]−1 at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site‐directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U(mg protein)−1, and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme‐substrate complexes suggested that Gln27 of wild‐type A. fumigatus phytase forms a hydrogen bond with the 6‐phosphate group of myo‐inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate‐limiting step of the A. fumigatus wild‐type phytase reaction.
The dihydrolipoyl acyltransferase (BCE2) subunit of the plant branched‐chain α‐ketoacid dehydrogenase complex forms a 24‐mer core with octagonal symmetry
Authors: Mooney Brian P. , Henzl Michael T. , Randall Douglas D. , Miernyk Jan A.
Published: February 2009

Little is known of the plant branched‐chain α‐ketoacid dehydrogenase complex. We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach. Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli. The soluble recombinant protein was purified using a Superose 6 size‐exclusion column to >90% homogeneity and was catalytically active. The recombinant protein formed a stable complex with a native molecular mass of 0.95 MDa and an S coefficient of 19.4, consistent with formation of a 24‐mer. Negative‐staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry. Despite divergence of mammalian and plant BCE2s, there is clearly conservation of structure that is independent of primary sequence.
Van't Hoff enthalpies without baselines
Authors: John Deborah M. , Weeks Kevin M.
Published: February 2009

Analysis of thermal melting curves represents one important approach for evaluating protein stability and the consequences of amino acid substitution on protein structure. By use of the van't Hoff relationship, the differential melting curve can be robustly fit to only three parameters, two of which are the underlying physical constants of melting temperature (Tm) and van't Hoff enthalpy (ΔHvH). Calculated Tm and ΔHvH values are insensitive to the choice of pre‐ and post‐transition baselines. Consequently, the method accurately computes Tm and ΔHvH for extremely truncated data sets, in the complete absence of baseline information, and for proteins with low melting temperatures, where the traditional direct approach routinely fails. Moreover, agreement between ΔHvH values obtained using points derived from pre‐vs. post‐transition data provide an independent method for detecting some classes of non‐two‐state transitions. Finally, fitting of the differential denaturation curve should prove useful for analysis of abbreviated data sets obtained from high throughput array analysis of protein stability.
Improving the quality of twilight‐zone alignments
Authors: Jaroszewski Lukasz , Godzik Adam , Rychlewski Leszek
Published: February 2009

Several recent publications illustrated advantages of using sequence profiles in recognizing distant homologies between proteins. At the same time, the practical usefulness of distant homology recognition depends not only on the sensitivity of the algorithm, but also on the quality of the alignment between a prediction target and the template from the database of known proteins. Here, we study this question for several supersensitive protein algorithms that were previously compared in their recognition sensitivity (Rychlewski et al., 2000). A database of protein pairs with similar structures, but low sequence similarity is used to rate the alignments obtained with several different methods, which included sequence–sequence, sequence–profile, and profile–profile alignment methods. We show that incorporation of evolutionary information encoded in sequence profiles into alignment calculation methods significantly increases the alignment accuracy, bringing them closer to the alignments obtained from structure comparison.

In general, alignment quality is correlated with recognition and alignment score significance. For every alignment method, alignments with statistically significant scores correlate with both correct structural templates and good quality alignments. At the same time, average alignment lengths differ in various methods, making the comparison between them difficult. For instance, the alignments obtained by FFAS, the profile–profile alignment algorithm developed in our group are always longer that the alignments obtained with the PSI‐BLAST algorithms. To address this problem, we develop methods to truncate or extend alignments to cover a specified percentage of protein lengths. In most cases, the elongation of the alignment by profile–profile methods is reasonable, adding fragments of similar structure. The examples of erroneous alignment are examined and it is shown that they can be identified based on the model quality.
NMR investigation of the interaction of the inhibitor protein Im9 with its partner DNase
Authors: Boetzel Ruth , Czisch Michael , Kaptein Robert , Hemmings Andrew M. , James Richard , Kleanthous Colin , Moore Geoffrey R.
Published: February 2009

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C‐terminal DNase domain. Double‐ and triple‐resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3–84 were 0.76 ± 0.14 Å for the backbone atoms and 1.36 ± 0.15 Å for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 Å, respectively, for residues 4–83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H‐1H‐13C NOESY‐HSQC spectra with and without 13C decoupling. The NMR‐defined interface agrees well with that determined in a recent X‐ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.
Thermodynamic dissection of the binding energetics of KNI‐272, a potent HIV‐1 protease inhibitor
Authors: Velazquez‐Campoy Adrian , Luque Irene , Todd Matthew J. , Milutinovich Mark , Kiso Yoshiaki , Freire Ernesto
Published: February 2009

KNI‐272 is a powerful HIV‐1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild‐type and drug‐resistant mutant V82F/I84V is presented. Unlike other protease inhibitors, KNI‐272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100–500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3–3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 × 1010 M−1 (Kd = 16 pM). Global analysis of the data yield a buffer‐ and pH‐independent binding enthalpy of −6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is −14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI‐272 is both enthalpically and entropically favorable. The structure‐based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI‐272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.
Structure‐based prediction of binding peptides to MHC class I molecules: Application to a broad range of MHC alleles
Authors: Schueler‐Furman Ora , Altuvia Yael , Sette Alessandro , Margalit Hanah
Published: February 2009

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T‐cells. Prediction of MHC‐binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T‐cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide‐MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623–644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361–369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.
Structure of streptococcal pyrogenic exotoxin A reveals a novel metal cluster
Authors: Earhart Cathleen A. , Vath Gregory M. , Roggiani Manuela , Schlievert Patrick M. , Ohlendorf Douglas H.
Published: February 2009

The streptococcal pyrogenic toxins A, B, and C (SPEA, SPEB, and SPEC) are responsible for the fever, rash, and other toxicities associated with scarlet fever and streptococcal toxic shock syndrome. This role, together with the ubiquity of diseases caused by Streptococcus pyogenes, have prompted structural analyses of SPEA by several groups. Papageorgiou et al. (1999) have recently reported the structure of SPEA crystallized in the absence of zinc. Zinc has been shown to be important in the ability of some staphylococcal and streptococcal toxins to stimulate proliferation of CD4+ T‐cells. Since cadmium is more electron dense than zinc and typically binds interchangeably, we grew crystals in the presence of 10 mM CdCl2. Crystals have been obtained in three space groups, and the structure in the P212121 crystal form has been refined to 1.9 Å resolution. The structural analysis revealed an identical tetramer as well as a novel tetrahedral cluster of cadmium in all three crystal forms on a disulfide loop encompassing residues 87–98. No cadmium was bound at the site homologous to the zinc site in staphylococcal enterotoxins C (SECs) despite the high structural homology between SPEA and SECs. Subsequent soaking of crystals grown in the presence of cadmium in 10 mM ZnCl2 showed that zinc binds in this site (indicating it can discriminate between zinc and cadmium ions) using the three ligands (Asp77, His106, and His 110) homologous to the SECs plus a fourth ligand (Glu33).
Identifying Organizational Drivers of Internal Audit Effectiveness
Authors: Arena Marika , Azzone Giovanni
Published: February 2009

This study attempts to understand the organizational drivers of internal audit effectiveness in the light of recent changes in the ‘mission’ of internal auditing and its central role in corporate governance. On the basis of data from 153 Italian companies, our survey shows that the effectiveness of internal auditing is influenced by: (1) the characteristics of the internal audit team, (2) the audit processes and activities, and (3) the organizational links. Internal audit effectiveness increases in particular when the ratio between the number of internal auditors and employees grows, the Chief Audit Executive is affiliated to the Institute of Internal Auditors, the company adopts control risk self‐assessment techniques, and the audit committee is involved in the activities of the internal auditors.
Review article: small intestinal bacterial overgrowth, bile acid malabsorption and gluten intolerance as possible causes of chronic watery diarrhoea
Authors: FAN X. , SELLIN J. H.
Published: February 2009

Background  Chronic watery diarrhoea is one of the most common symptoms prompting GI evaluation. Recently, new diagnostic considerations have emerged as possible factors in chronic diarrhoea.

Aim  To review available data regarding diagnosis and treatment of chronic diarrhoea with an emphasis on bacterial overgrowth and bile acid malabsorption.

Methods  A systematic search of the English language literature of chronic diarrhoea was performed focused on three possible aetiologies of diarrhoea: small intestinal bacterial overgrowth (SIBO), idiopathic bile salt malabsorption (IBAM), gluten responsive enteropathy.

Results  Recent studies suggest that SIBO and bile acid malabsorption may have been underestimated as possible causes of chronic watery diarrhoea. Gluten intolerance with negative coeliac serology is a contentious possible cause of watery diarrhoea, but requires further research before acceptance as an entity.

Conclusion  In patients with otherwise unexplained chronic watery diarrhoea, small intestinal bacterial overgrowth and bile salt malabsorption should be considered and investigated.