Registering alpha-helices and beta-strands using backbone C-H...O interactions

The possible occurrence of a novel helix terminating structural motif in proteins involving a stabilizing short C-H...O interaction has been examined using a dataset of 634 non-homologous protein structures (相似文献   

An unusual C-H...O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix

An unusual C-terminal conformation has been detected in a synthetic decapeptide designed to analyze the stereochemistry of helix termination in polypeptides. The crystal structure of the decapeptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe reveals a helical segment spanning residues 1-7 and helix termination by formation of a Schellman motif, generated by (D)Ala(8) adopting the left-handed helical (alpha(L)) conformation. The extended conformation at (D)Leu(9) results in a compact folded structure, stabilized by a potentially strong C-H. O hydrogen bond between Ala(4) C(alpha)H and (D)Leu(9) CO. The parameters for C-H. O interaction are Ala(4) C(alpha)H. O=C (D)Leu(9) distance 3.27 A, C(alpha)-H. O angle 176 degrees, and O. H(alpha) distance 2.29 A. This structure suggests that insertion of contiguous D-residues may provide a handle for the generation of designed structures containing more than one helical segment folded in a compact manner.     

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.     

Conformational features of a hexapeptide model Ac-TGAAKA-NH2 corresponding to a hydrated alpha helical segment from glyceraldehyde 3-phosphate dehydrogenase: implications for the role of turns in helix folding

Recent analysis of alpha helices in protein crystal structures, available in literature, revealed hydrated alpha helical segments in which, water molecule breaks open helix 5-->1 hydrogen bond by inserting itself, hydrogen bonds to both C=O and NH groups of helix hydrogen bond without disrupting the helix hydrogen bond, and hydrogen bonds to either C=O or NH of helix hydrogen bond. These hydrated segments display a variety of turn conformations and are thought to be 'folding intermediates' trapped during folding-unfolding of alpha helices. A role for reverse turns is implicated in the folding of alpha helices. We considered a hexapeptide model Ac-1TGAAKA6-NH2 from glyceraldehyde 3-phosphate dehydrogenase, corresponding to a hydrated helical segment to assess its role in helix folding. The sequence is a site for two 'folding intermediates'. The conformational features of the model peptide have been investigated by 1H 2D NMR techniques and quantum mechanical perturbative configuration interaction over localized orbitals (PCILO) method. Theoretical modeling largely correlates with experimental observations. Based upon the amide proton temperature coefficients, the observed d alpha n(i, i + 1), d alpha n(i, i + 2), dnn(i, i + 1), d beta n(i, i + 1) NOEs and the results from theoretical modeling, we conclude that the residues of the peptide sample alpha helical and neck regions of the Ramachandran phi, psi map with reduced conformational entropy and there is a potential for turn conformations at N and C terminal ends of the peptide. The role of reduced conformational entropy and turn potential in helix formation have been discussed. We conclude that the peptide sequence can serve as a 'folding intermediate' in the helix folding of glyceraldehyde 3-phosphate dehydrogenase.     

The occurrence of C--H...O hydrogen bonds in alpha-helices and helix termini in globular proteins

A comprehensive database analysis of C--H...O hydrogen bonds in 3124 alpha-helices and their corresponding helix termini has been carried out from a nonredundant data set of high-resolution globular protein structures resolved at better than 2.0 A in order to investigate their role in the helix, the important protein secondary structural element. The possible occurrence of 5 --> 1 C--H...O hydrogen bond between the ith residue CH group and (i - 4)th residue C==O with C...O < or = 3.8 A is studied, considering as potential donors the main-chain Calpha and the side-chain carbon atoms Cbeta, Cgamma, Cdelta and Cepsilon. Similar analysis has been carried out for 4 --> 1 C--H...O hydrogen bonds, since the C--H...O hydrogen bonds found in helices are predominantly of type 5 --> 1 or 4 --> 1. A total of 17,367 (9310 of type 5 --> 1 and 8057 of type 4 --> 1) C--H...O hydrogen bonds are found to satisfy the selected criteria. The average stereochemical parameters for the data set suggest that the observed C--H...O hydrogen bonds are attractive interactions. Our analysis reveals that the Cgamma and Cbeta hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic beta-branched residue Ile to participate in 5 --> 1 C--H...O hydrogen bonds involving methylene Cgamma 1 atom as donor in alpha-helices. This may be an enthalpic compensation for the greater loss of side-chain conformational entropy for beta-branched amino acids due to the constraint on side-chain torsion angle, namely, chi1, when they occur in helices. The preference of amino acids for 4 --> 1 C--H...O hydrogen bonds is found to be more for Asp, Cys, and for aromatic residues Trp, Phe, and His. Interestingly, overall propensity for C--H...O hydrogen bonds shows that a majority of the helix favoring residues such as Met, Glu, Arg, Lys, Leu, and Gln, which also have large side-chains, prefer to be involved in such types of weak attractive interactions in helices. The amino acid side-chains that participate in C--H...O interactions are found to shield the acceptor carbonyl oxygen atom from the solvent. In addition, C--H...O hydrogen bonds are present along with helix stabilizing salt bridges. A novel helix terminating interaction motif, X-Gly with Gly at C(cap) position having 5 --> 1 Calpha--H...O, and a chain reversal structural motif having 1 --> 5 Calpha-H...O have been identified and discussed. Our analysis highlights that a multitude of local C--H...O hydrogen bonds formed by a variety of amino acid side-chains and Calpha hydrogen atoms occur in helices and more so at the helix termini. It may be surmised that the main-chain Calpha and the side-chain CH that participate in C--H...O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N--H...O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.     

Stabilization of a novel beta-turn-like motif by nonconventional intramolecular hydrogen-bonding interactions in a model peptide incorporating beta-alanine

The chemical synthesis and x-ray crystal structure analysis of a model peptide incorporating a conformationally adaptable unsubstituted beta-Ala residue: Boc-beta-Ala-Acc6-OCH3 (C16H28N2O5, molecular weight = 328.41; 1) has been described. The peptide crystallized in the space group P2(1)2(1)2(1) a = 8.537 (3), b = 8.872 (10), c = 25.327 (8), alpha = beta = gamma = 90.0 degrees, Z = 4. An attractive feature of the crystal structure analysis of 1 is an accommodation of a significantly folded beta-Ala residue in a short linear peptide. The overall peptide conformation is typically folded into a beta-turn-like motif. The stabilization of the peptide backbone conformation by nonconventional C-H...O weak intramolecular hydrogen-bonding interactions, involving the ester terminal carbon atom and the ethereal oxygen of the Boc group, has been evoked. The conformational constraint that seems most apparent is the phi, psi value of the highly constrained hydrophobic Acc6 ring that may play a key role in inducing or sustaining the observed pseudo type III or III' beta-turn structure. The resulting 12-membered hydrogen bonding ring motif in 1 is distinctly different from the one found in classical beta-turn structures, stabilized by a conventional strong C=O...H-N intramolecular hydrogen bond, comprised of alpha-amino acids. The potential of the conformationally adaptable beta-Ala residue to occupy i + 1 position (left corner) of the folded beta-turn-like structure and to design and construct novel secondary structural features have been emphasized.     

Cooperative effects in hydrogen-bonding of protein secondary structure elements: a systematic analysis of crystal data using Secbase

A systematic analysis of the hydrogen-bonding geometry in helices and beta sheets has been performed. The distances and angles between the backbone carbonyl O and amide N atoms were correlated considering more than 1500 protein chains in crystal structures determined to a resolution better than 1.5 A. They reveal statistically significant trends in the H-bond geometry across the different secondary structural elements. The analysis has been performed using Secbase, a modular extension of Relibase (Receptor Ligand Database) which integrates information about secondary structural elements assigned to individual protein structures with the various search facilities implemented into Relibase. A comparison of the mean hydrogen-bond distances in alpha helices and 3(10) helices of increasing length shows opposing trends. Whereas in alpha helices the mean H-bond distance shrinks with increasing helix length and turn number, the corresponding mean dimension in 3(10) helices expands in a comparable series. Comparing similarly the hydrogen-bond lengths in beta sheets there is no difference to be found between the mean H-bond length in antiparallel and parallel beta sheets along the strand direction. In contrast, an interesting systematic trend appears to be given for the hydrogen bonds perpendicular to the strands bridging across an extended sheet. With increasing number of accumulated strands, which results in a growing number of back-to-back piling hydrogen bonds across the strands, a slight decrease of the mean H-bond distance is apparent in parallel beta sheets whereas such trends are obviously not given in antiparallel beta sheets. This observation suggests that cooperative effects mutually polarizing spatially well-aligned hydrogen bonds are present either in alpha helices and parallel beta sheets whereas such influences seem to be lacking in 3(10) helices and antiparallel beta sheets.     

Peptide design: Crystal structure of a helical peptide module attached to a potentially nonhelical amino terminal segment

The peptide Boc-Gly-Dpg-Gly-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe has been designed to examine the structural consequences of placing a short segment with a low helix propensity at the amino terminus of a helical heptapeptide module. The Gly-Dpg-Gly segment is a potential connecting element in the synthetic construction of a helix-linker-helix motif. Crystal parameters for the peptide are P2₁, a = 8.651(3) Å, b = 46.826(13) Å, c = 16.245 Å, β = 90.13(3)*, Z = 4; 2 independent molecules/asymmetric unit. The structure reveals almost identical conformations for the two independent molecules. The backbone is completely helical for residues 2–9, with one 4 → 1 hydrogen bond and six 5 → 1 hydrogen bonds. The α,α-di-n-propylglycine residue adopts a helical conformation. Gly(1) adopts an extended conformation resulting in a nonhelical N-terminus, with the Boc group swinging away from the helix. The lateral association of helices in the b axis direction is unusual in that the helix axes are directed up or down (parallel or antiparallel) by pairs: ↓↓↑↑↓↓, etc. © 1996 John Wiley & Sons, Inc.     

Stereochemistry of Schellman motifs in peptides: Crystal structure of a hexapeptide with a C‐terminus 6 → 1 hydrogen bond

The Schellman motif is a widely observed helix terminating structural motif in proteins, which is generated when the C‐terminus residue adopts a left‐handed helical (α_L) conformation. The resulting hydrogen‐bonding pattern involves the formation of an intramolecular 6 → 1 interaction. This helix terminating motif is readily mimicked in synthetic helical peptides by placing an achiral residue at the penultimate position of the sequence. Thus far, the Schellman motif has been characterized crystallographically only in peptide helices of length 7 residues or greater. The structure of the hexapeptide Boc–Pro–Aib–Gly–Leu–Aib–Leu–OMe in crystals reveal a short helical stretch terminated by a Schellman motif, with the formation of 6 → 1 C‐terminus hydrogen bond. The crystals are in the space group P2₁2₁2₁ with a = 18.155(3) Å, b = 18.864(8) Å, c = 11.834(4) Å, and Z = 4 . The final R₁ and wR₂ values are 7.68 and 14.6%, respectively , for 1524 observed reflections [F_o ≥ 3ς(F_o)]. A 6 → 1 hydrogen bond between Pro(1)CO · · · Leu(6)NH and a 5 → 2 hydrogen bond between Aib(2)CO · · · Aib(5)NH are observed. An analysis of the available oligopeptides having an achiral Aib residue at the penultimate position suggests that chain length and sequence effects may be the other determining factors in formation of Schellman motifs. © 1999 John Wiley & Sons, Inc. Biopoly 50: 13–22, 1999     

Controls exerted by the Aib residue: helix formation and helix reversal

The helix forming properties of the achiral alpha-amino isobutyric residue (Aib) have been demonstrated by numerous crystal structure analyses of designed and naturally occurring peptides containing one or more Aib residues in the sequence. Experimental and computational results concerning the type of helix obtained, whether the 3(10)-helix with 4 --> 1 type hydrogen bonds or the alpha-helix with 5 --> 1 hydrogen bonds or mixtures of the two, have been published. This paper deals with residues that, if inserted into a sequence, could perturb the helix-forming propensity afforded by the presence of Aib residues. Examples of structures will be presented in which Pro, Hyp, Gly-Gly, d-Ala-Gly, and Lac have been centrally placed in the sequence. In addition to the formation of helices, detailed experimentally obtained conformation information is presented for the role of the Aib residue in reversing the sense of the helix (the Schellman motif) with the consequent formation of the 6 --> 1 type hydrogen bond or a solvated 6 --> 1 hydrogen bond. Data are presented for 13 molecules with helix reversals at the C-terminus or near the center of the sequence.     

Evaluation of C-H cdots,three dots,centered O hydrogen bonds in native and misfolded proteins

Non-traditional C-H cdots, three dots, centered Y hydrogen bonds, in which a carbon atom acts as the hydrogen donor and an electronegative atom Y (Y=N, O or S) acts as the acceptor, have been reported in proteins, but their importance in protein structures is not well established. Here, we present the results of three computational tests that examine the significance of C-H cdots, three dots, centered Y bonds involving the C(alpha) in proteins. First, we compared the number of C(alpha)-H cdots, three dots, centered Y bonds in native structures with two sets of compact, energy-minimized decoy structures. The decoy structures contain about as many C(alpha)-H cdots, three dots, centered Y bonds as the native structures, indicating that the constraints of chain connectivity and compactness can lead to incidental formation of C(alpha)-H cdots, three dots, centered Y bonds. Secondly, we examined whether short C(alpha)-H cdots, three dots, centered Y bonds have a tendency to be linear, as is expected for a cohesive hydrogen-bonding interaction. The native structures do show this trend, but so does one of the decoy sets, suggesting that this criterion is also not sufficient to indicate a stabilizing interaction. Finally, we examined the preference for C(alpha)-H cdots, three dots, centered Y bond donors to be near to strong hydrogen bond acceptors. In the native proteins, the alpha protons attract strong acceptors like oxygen atoms more than weak acceptors. In contrast, hydrogen bond donors in the decoy structures do not distinguish between strong and weak acceptors. Thus, any individual C(alpha)-H cdots, three dots, centered Y bond may be fortuitous and occur due to the polypeptide connectivity and compactness. Taken collectively, however, C(alpha)-H cdots, three dots, centered Y bonds provide a weakly cohesive force that stabilizes proteins.     

Synthetic peptide helices in crystals: structure and antiparallel and skewed packing motifs for alpha-helices in two isomeric decapeptides

The isomeric decapeptides Boc-Aib-Ala-Leu-Ala-Aib-Aib-Leu-Ala-Leu-Aib-OMe (II) and Boc-Aib-Ala-Aib-Ala-Leu-Ala-Leu-Aib-Leu-Aib-OMe (III), are predominantly alpha-helical with little effect on the conformation with interchange of Aib/Ala residues or Aib/Leu residues. The packing motif of helices in crystal II is antiparallel, whereas the helices pack in a skewed fashion in crystal III, with a 40 degrees angle between neighboring helix axes. Crystal III contains a water molecule in a hydrophobic hole that forms hydrogen bonds with two carbonyl oxygens that also participate in 5----1 type hydrogen bonds. Values for helical torsional angles phi and psi assume a much wider range than anticipated. Crystal II: C49H88N10O13, space group P2(1), with a = 16.625 (2) A, b = 9.811 (5) A, c = 18.412 (3) A, beta = 99.79 (1) degrees, Z = 2, R = 5.7% for 4338 data with magnitude of F0' greater than 3 sigma(F). Crystal III: C49H88N10O13 x 1/2H2O, space group P2(1) with a = 11.072 (2) A, b = 34.663 (5) A, c = 16.446 (3) A, beta = 107.85 (1) degrees, Z = 4, R = 8.3% for 6087 data with [F0[ greater than 3 sigma(F).     

The crystal structure of the alpha-cellobiose.2 NaI.2 H(2)O complex in the context of related structures and conformational analysis

The crystal structure of beta-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranose (alpha-cellobiose) in a complex with water and NaI was determined with Mo K(alpha) radiation at 150 K to R=0.027. The space group is P2(1) and unit cell dimensions are a=9.0188, b=12.2536, c=10.9016 A, beta=97.162 degrees. There are no direct hydrogen bonds among cellobiose molecules, and the usual intramolecular hydrogen bond between O-3 and O-5' is replaced by a bridge involving Na+, O-3, O-5', and O-6'. Both Na+ have sixfold coordination. One I(-) accepts six donor hydroxyl groups and three C-H***I(-) hydrogen bonds. The other accepts three hydroxyls, one Na+, and five C-H***I(-) hydrogen bonds. Linkage torsion angles phi(O-5) and psi(C-5) are -73.6 and -105.3 degrees, respectively (phi(H)=47.1 degrees and psi(H)=14.6 degrees ), probably induced by the Na+ bridge. This conformation is in a separate cluster in phi,psi space from most similar linkages. Both C-6-O-H and C-6'-O-H are gg, while the C-6'-O-H groups from molecules not in the cluster have gt conformations. Hybrid molecular mechanics/quantum mechanics calculations show <1.2 kcal/mol strain for any of the small-molecule structures. Extrapolation of the NaI cellobiose geometry to a cellulose molecule gives a left-handed helix with 2.9 residues per turn. The energy map and small-molecule crystal structures imply that cellulose helices having 2.5 and 3.0 residues per turn are left-handed.     

Parallel and antiparallel aggregation of alpha-helices. Crystal structures of two apolar decapeptides X-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe (X = Boc, Ac)

An apolar helical decapeptide with different end groups, Boc- or Ac-, crystallizes in a completely parallel fashion for the Boc-analog and in an antiparallel fashion for the Ac-analog. In both crystals, the packing motif consists of rows of parallel molecules. In the Boc-crystals, adjacent rows assemble with the helix axes pointed in the same direction. In the Ac-crystals, adjacent rows assemble with the helix axes pointed in opposite directions. The conformations of the molecules in both crystals are quite similar, predominantly alpha-helical, except for the tryptophanyl side chain where chi 1 congruent to 60 degrees in the Boc- analog and congruent to 180 degrees in the Ac-analog. As a result, there is one lateral hydrogen bond between helices, N(1 epsilon)...O(7), in the Ac-analog. The structures do not provide a ready rationalization of packing preference in terms of side-chain interactions and do not support a major role for helix dipole interactions in determining helix orientation in crystals. The crystal parameters are as follow. Boc-analog: C60H97N11O13.C3H7OH, space group Pl with a = 10.250(3) A, b = 12.451(4) A, c = 15.077(6) A, alpha = 96.55(3) degrees, beta = 92.31(3) degrees, gamma = 106.37(3) degrees, Z = 1, R = 5.5% for 5581 data ([F] greater than 3.0 sigma(F)), resolution 0.89 A. Ac-analog: C57H91N11O12, space group P2(1) with a = 9.965(1) A, b = 19.707(3) A, c = 16.648(3) A, beta = 94.08(1), Z = 2, R = 7.2% for 2530 data ([F] greater than 3.0 sigma(F)), resolution 1.00 A.     

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.     

Unfolding of an alpha-helix in water.

We describe a 1 ns molecular dynamics simulation of an 18-residue peptide (corresponding to a portion of the H helix of myoglobin) in water. The initial helical conformation progressively frays to a more disordered structure, with the loss of internal secondary structure generally proceeding from the C-terminus toward the N-terminus. Although a variety of mechanisms are involved in the breaking of helical hydrogen bonds, the formation of transient turn structures, with i----i + 3 hydrogen bonds, and bifurcated hydrogen-bond structures intermediate between alpha and turn or 3(10) structures is a common motif. In some cases a single water molecule is inserted into an internal hydrogen bond, but it is also common to have several water molecules involved in transient intermediates. Overall, the results provide new information about the detailed mechanisms by which helices are made and broken in aqueous solution.     

Stereospecific interactions of proline residues in protein structures and complexes

The constrained backbone torsion angle of a proline (Pro) residue has usually been invoked to explain its three-dimensional context in proteins. Here we show that specific interactions involving the pyrrolidine ring atoms also contribute to its location in a given secondary structure and its binding to another molecule. It is adept at participating in two rather non-conventional interactions, C-H...pi and C-H...O. The geometry of interaction between the pyrrolidine and aromatic rings, vis-à-vis the occurrence of the C-H...pi interactions has been elucidated. Some of the secondary structural elements stabilized by Pro-aromatic interactions are beta-turns, where a Pro can interact with an adjacent aromatic residue, and in antiparallel beta-sheet, where a Pro in an edge strand can interact with an aromatic residue in the adjacent strand at a non-hydrogen-bonded site. The C-H groups at the Calpha and Cdelta positions can form strong C-H...O interactions (as seen from the clustering of points) and such interactions involving a Pro residue at C' position relative to an alpha-helix can cap the hydrogen bond forming potentials of the free carbonyl groups at the helix C terminus. Functionally important Pro residues occurring at the binding site of a protein almost invariably engage aromatic residues (with one of them being held by C-H...pi interaction) from the partner molecule in the complex, and such aromatic residues are highly conserved during evolution.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Studies of the erythrocyte spectrin tetramerization region

Human erythrocyte spectrin dimers associate at the N-terminal region of alpha spectrin (alpha N) and the C-terminal region of beta-spectrin (beta C) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of alpha-spectrin (Sp alpha 1-156 and Sp alpha 1-368, respectively), and found that both peptides associate with a beta-spectrin model peptide, with an affinity similar to that found in alpha beta dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in alpha-spectrin is helical even in the absence of its beta-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Sp alpha 1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Sp alpha-156 associates with helices in the beta peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Sp alpha 1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its beta-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of alpha-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T. A comparison of the structures of Sp alpha 1-156 and Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T is discussed.     

Pex, analytical tools for PDB files. II. H-Pex: noncanonical H-bonds in alpha-helices

We use the H-Pex (Thomas et al., this issue) to analyze the main chain interactions in 131 proteins. In antiparallel beta-sheets, the geometry of the N...O bond is: median N...O distances, 2.9 SA, C==O...N angles at 154 degrees and the C alpha--C==O...H angles are dispersed around 3 degrees. In some instances, the other side of the C==O axis is occupied by a HC alpha. As recently supported by Vargas et al. (J Am Chem Soc 2000;122:4750-4755) C alpha H...O and NH...O could cooperate to sheet stability. In alpha-helices, the main chain C==O interact with the NH of their n + 4 neighbor on one side, and with a C beta H or C gamma H on the other side. The median O...N distance (3.0 A) and C==N angle (147 degrees) suggest a canonical H-bond, but the C alpha--C==O...H dihedral angle invalidates this option, since the hydrogen attacks the oxygen at 122 degrees, i.e., between the sp(2) and pi orbitals. This supports that the H-bond is noncanonical. In many instances, the C gamma H or the C beta H of the n + 4 residue stands opposite to the NH with respect to the oxygen. Therefore, we propose that, in alpha-helices, the C gamma H or C beta H and the NH of the n + 4 residue hold the oxygen like an electrostatic pincher. Proteins 2001;43:37-44.