The energetics of off-rotamer protein side-chain conformations

Non-rotameric ("off-rotamer") conformations are commonly observed for the side-chains of protein crystal structures. This study examines whether such conformations are real or artifactual by comparing the energetics of on and off-rotamer side-chain conformations calculated with the CHARMM energy function. Energy-based predictions of side-chain orientation are carried out by rigid-geometry mapping in the presence of the fixed protein environment for 1709 non-polar side-chains in 24 proteins for which high-resolution (2.0 A or better) structures are available. For on-rotamer conformations, 97.6 % are correctly predicted; i.e. they correspond to the absolute minima of their local side-chain energy maps (generally to within 10 degrees or less). By contrast, for the observed off-rotamer side-chain conformations, 63.8 % are predicted correctly. This difference is statistically significant (P<0.001) and suggests that while most of the observed off-rotamer conformations are real, many of the erroneously predicted ones are likely to be artifacts of the X-ray refinements. Probabilities for off-rotamer conformations of the non-polar side-chains are calculated to be 5.0-6.1 % by adaptive umbrella-sampled molecular dynamics trajectories of individual amino acid residues in vacuum and in the presence of an average protein or aqueous dielectric environment. These results correspond closely to the 5.7 % off-rotamer fraction predicted by the rigid-geometry mapping studies. Since these values are about one-half of the 10.2 % off-rotamer fraction observed in the X-ray structures, they support the conclusion that many of the latter are artifacts. In both the rigid-geometry mapping and the molecular dynamics studies, the discrepancies between the predicted and observed fractions of off-rotamer conformations are largest for leucine residues (approximately 6 % versus 16.6 %). The simulations for the isolated amino acid residues indicate that the real off-rotamer frequency of 5-6 % is consistent with the internal side-chain and local side-chain-backbone energetics and does not originate from shifts due to the protein. The present results suggest that energy-based rotation maps can be used to find side-chain positional artifacts that appear in crystal structures based on refinements in the 2 A resolution range.     

Improved side-chain modeling for protein-protein docking

Success in high-resolution protein-protein docking requires accurate modeling of side-chain conformations at the interface. Most current methods either leave side chains fixed in the conformations observed in the unbound protein structures or allow the side chains to sample a set of discrete rotamer conformations. Here we describe a rapid and efficient method for sampling off-rotamer side-chain conformations by torsion space minimization during protein-protein docking starting from discrete rotamer libraries supplemented with side-chain conformations taken from the unbound structures, and show that the new method improves side-chain modeling and increases the energetic discrimination between good and bad models. Analysis of the distribution of side-chain interaction energies within and between the two protein partners shows that the new method leads to more native-like distributions of interaction energies and that the neglect of side-chain entropy produces a small but measurable increase in the number of residues whose interaction energy cannot compensate for the entropic cost of side-chain freezing at the interface. The power of the method is highlighted by a number of predictions of unprecedented accuracy in the recent CAPRI (Critical Assessment of PRedicted Interactions) blind test of protein-protein docking methods.     

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.     

Assessing the role of tryptophan residues in the binding site

Instead of looking at the interfacial area as a measure of the extent of a protein--protein recognition site, a new procedure has been developed to identify the importance of a specific residue, namely tryptophan, in the binding process. Trp residues which contribute more towards the free energy of binding have their accessible surface area reduced, on complex formation, for both the main-chain and side-chain atoms, whereas for the less important residues the reduction is restricted only to the aromatic ring of the side chain. The two categories of residues are also distinguished by the presence or absence of hydrogen bonds involving the Trp residue in the complex. A comparison of the observed change in the accessible surface area with the value calculated using an analytical expression provides another way of characterizing the Trp residues critical for binding and this has been used to identify such residues involved in binding non-proteinaceous molecules in protein structures.     

Occurrence of bifurcated three-center hydrogen bonds in proteins

Analysis of 13 high-resolution protein X-ray crystal structures shows that 1204 (24%) of all the 4974 hydrogen bonds are of the bifurcated three-center type with the donor X-H opposing two acceptors A1, A2. They occur systematically in alpha-helices where 90% of the hydrogen bonds are of this type; the major component is (n + 4)N-H ... O = C(n) as expected for a 3.6(13) alpha-helix, and the minor component is (n + 4)N-H ... O = C(n + 1), as observed in 3(10) helices; distortions at the C-termini of alpha-helices are stabilized by three-center bonds. In beta-sheets 40% of the hydrogen bonds are three-centered. The frequent occurrence of three-center hydrogen bonds suggests that they should not be neglected in protein structural studies.     

High-resolution solution structure of reduced French bean plastocyanin and comparison with the crystal structure of poplar plastocyanin

The three-dimensional solution structure of reduced (CuI) plastocyanin from French bean leaves has been determined by distance geometry and restrained molecular dynamics methods using constraints obtained from 1H n.m.r. (nuclear magnetic resonance) spectroscopy. A total of 1244 experimental constraints were used, including 1120 distance constraints, 103 dihedral angle constraints and 21 hydrogen bond constraints. Stereospecific assignments were made for 26 methylene groups and the methyls of 11 valines. Additional constraints on copper co-ordination were included in the restrained dynamics calculations. The structures are well defined with average atomic root-mean-square deviations from the mean of 0.45 A for all backbone heavy atoms and 1.08 A for side-chain heavy atoms. French bean plastocyanin adopts a beta-sandwich structure in solution that is similar to the X-ray structure of reduced poplar plastocyanin; the average atomic root-mean-square difference between 16 n.m.r. structures and the X-ray structure is 0.76 A for all backbone heavy atoms. The conformations of the side-chains that constitute the hydrophobic core of French bean plastocyanin are very well defined. Of 47 conserved residues that populate a single chi 1 angle in solution, 43 have the same rotamer in the X-ray structure. Many surface side-chains adopt highly preferred conformations in solution, although the 3J alpha beta coupling constants often indicate some degree of conformational averaging. Some surface side-chains are disordered in both the solution and crystal structures of plastocyanin. There is a striking correlation between measures of side-chain disorder in solution and side-chain temperature factors in the X-ray structure. Side-chains that form a distinctive acidic surface region, believed to be important in binding other electron transfer proteins, appear to be disordered. Fifty backbone amide protons form hydrogen bonds to carbonyls in more than 60% of the n.m.r. structures; 45 of these amide protons exchange slowly with solvent deuterons. Ten hydrogen bonds are formed between side-chain and backbone atoms, eight of which are correlated with decreased proton exchange. Of the 60 hydrogen bonds formed in French bean plastocyanin, 56 occur in the X-ray structure of the poplar protein; two of the missing hydrogen bonds are absent as a result of mutations. It appears that molecular dynamics refinement of highly constrained n.m.r. structures allows accurate prediction of the pattern of hydrogen bonding.     

The dominant role of side-chain backbone interactions in structural realization of amino acid code. ChiRotor: a side-chain prediction algorithm based on side-chain backbone interactions

The basic differences between the 20 natural amino acid residues are due to differences in their side-chain structures. This characteristic design of protein building blocks implies that side-chain-side-chain interactions play an important, even dominant role in 3D-structural realization of amino acid codes. Here we present the results of a comparative analysis of the contributions of side-chain-side-chain (s-s) and side-chain-backbone (s-b) interactions to the stabilization of folded protein structures within the framework of the CHARMm molecular data model. Contrary to intuition, our results suggest that side-chain-backbone interactions play the major role in side-chain packing, in stabilizing the folded structures, and in differentiating the folded structures from the unfolded or misfolded structures, while the interactions between side chains have a secondary effect. An additional analysis of electrostatic energies suggests that combinatorial dominance of the interactions between opposite charges makes the electrostatic interactions act as an unspecific folding force that stabilizes not only native structure, but also compact random conformations. This observation is in agreement with experimental findings that, in the denatured state, the charge-charge interactions stabilize more compact conformations. Taking advantage of the dominant role of side-chain-backbone interactions in side-chain packing to reduce the combinatorial problem, we developed a new algorithm, ChiRotor, for rapid prediction of side-chain conformations. We present the results of a validation study of the method based on a set of high resolution X-ray structures.     

Deterministic features of side-chain main-chain hydrogen bonds in globular protein structures

A total of 19 835 polar residues from a data set of 250 non-homologous and highly resolved protein crystal structures were used to identify side-chain main-chain (SC-MC) hydrogen bonds. The ratio of the number of SC-MC hydrogen bonds to the total number of polar residues is close to 1:2, indicating the ubiquitous nature of such hydrogen bonds. Close to 56% of the SC-MC hydrogen bonds are local involving side-chain acceptor/donor ('i') and a main-chain donor/acceptor within the window i-5 to i+5. These short-range hydrogen bonds form well defined conformational motifs characterized by specific combinations of backbone and side-chain torsion angles. (a) The Ser/Thr residues show the greatest preference in forming intra-helical hydrogen bonds between the atoms O(gamma)(i) and O(i-4). More than half the examples of such hydrogen bonds are found at the middle of alpha-helices rather than at their ends. The most favoured motif of these examples is alpha(R)alpha(R)alpha(R)alpha(R)(g(-)). (b) These residues also show great preference to form hydrogen bonds between O(gamma)(i) and O(i-3), which are closely related to the previous type and though intra-helical, these hydrogen bonds are more often found at the C-termini of helices than at the middle. The motif represented by alpha(R)alpha(R)alpha(R)alpha(R)(g(+)) is most preferred in these cases. (c) The Ser, Thr and Glu are the most frequently found residues participating in intra-residue hydrogen bonds (between the side-chain and main-chain of the same residue) which are characterized by specific motifs of the form beta(g(+)) for Ser/Thr residues and alpha(R)(g(-)g(+)t) for Glu/Gln. (d) The side-chain acceptor atoms of Asn/Asp and Ser/Thr residues show high preference to form hydrogen bonds with acceptors two residues ahead in the chain, which are characterized by the motifs beta (tt')alphaR and beta(t)alpha(R), respectively. These hydrogen bonded segments, referred to as Asx turns, are known to provide stability to type I and type I' beta-turns. (e) Ser/Thr residues often form a combination of SC-MC hydrogen bonds, with the side-chain donor hydrogen bonded to the carbonyl oxygen of its own peptide backbone and the side-chain acceptor hydrogen bonded to an amide hydrogen three residues ahead in the sequence. Such motifs are quite often seen at the beginning of alpha-helices, which are characterized by the beta(g(+))alpha(R)alpha(R) motif. A remarkable majority of all these hydrogen bonds are buried from the protein surface, away from the surrounding solvent. This strongly indicates the possibility of side-chains playing the role of the backbone, in the protein interiors, to satisfy the potential hydrogen bonding sites and maintaining the network of hydrogen bonds which is crucial to the structure of the protein.     

Positioning hydrogen atoms by optimizing hydrogen-bond networks in protein structures

A method is presented that positions polar hydrogen atoms in protein structures by optimizing the total hydrogen bond energy. For this goal, an empirical hydrogen bond force field was derived from small molecule crystal structures. Bifurcated hydrogen bonds are taken into account. The procedure also predicts ionization states of His, Asp, and Glu residues. During optimization, sidechain conformations of His, Gln, and Asn residues are allowed to change their last χ angle by 180° to compensate for crystallographic misassignments. Crystal structure symmetry is taken into account where appropriate. The results can have significant implications for molecular dynamics simulations, protein engineering, and docking studies. The largest impact, however, is in protein structure verification: over 85% of protein structures tested can be improved by using our procedure. Proteins 26:363–376 © 1996 Wiley-Liss, Inc.     

Are aromatic carbon donor hydrogen bonds linear in proteins?

Proteins fold and maintain structure through the collective contributions of a large number of weak, noncovalent interactions. The hydrogen bond is one important category of forces that acts on very short distances. As our knowledge of protein structure continues to expand, we are beginning to appreciate the role that weak carbon-donor hydrogen bonds play in structure and function. One property that differentiates hydrogen bonds from other packing forces is propensity for forming a linear donor-hydrogen-acceptor orientation. To ascertain if carbon-donor hydrogen bonds are able to direct acceptor linearity, we surveyed the geometry of interactions specifically involving aromatic sidechain ring carbons in a data set of high resolution protein structures. We found that while donor-acceptor distances for most carbon donor hydrogen bonds were tighter than expected for van der Waals packing, only the carbons of histidine showed a significant bias for linear geometry. By categorizing histidines in the data set into charged and neutral sidechains, we found only the charged subset of histidines participated in linear interactions. B3LYP/6-31G**++ level optimizations of imidazole and indole-water interactions at various fixed angles demonstrates a clear orientation dependence of hydrogen bonding capacity for both charged and neutral sidechains. We suggest that while all aromatic carbons can participate in hydrogen bonding, only charged histidines are able to overcome protein packing forces and enforce linear interactions. The implications for protein modeling and design are discussed.     

Analysis of a data set of paired uncomplexed protein structures: new metrics for side-chain flexibility and model evaluation

We compiled and analyzed a data set of paired protein structures containing proteins for which multiple high-quality uncomplexed atomic structures were available in the Protein Data Bank. Side-chain flexibility was quantified, yielding a set of residue- and environment-specific confidence levels describing the range of motion around chi1 and chi2 angles. As expected, buried residues were inflexible, adopting similar conformations in different crystal structure analyses. Ile, Thr, Asn, Asp, and the large aromatics also showed limited flexibility when exposed on the protein surface, whereas exposed Ser, Lys, Arg, Met, Gln, and Glu residues were very flexible. This information is different from and complementary to the information available from rotamer surveys. The confidence levels are useful for assessing the significance of observed side-chain motion and estimating the extent of side-chain motion in protein structure prediction. We compare the performance of a simple 40 degrees threshold with these quantitative confidence levels in a critical evaluation of side-chain prediction with the program SCWRL.     

Structure of scorpion toxin variant-3 at 1.2 A resolution.

The crystal structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been refined at 1.2 A resolution using restrained least-squares. The final model includes 492 non-hydrogen protein atoms, 453 protein hydrogen atoms, eight 2-methyl-2,4-pentanediol (MPD) solvent atoms, and 125 water oxygen atoms. The variant-3 protein model geometry deviates from ideal bond lengths by 0.024 A and from ideal angles by 3.6 degrees. The crystallographic R-factor for structure factors calculated from the final model is 0.192 for 17,706 unique reflections between 10.0 to 1.2 A. A comparison between the models of the initial 1.8 A and the 1.2 A refinement shows a new arrangement of the previously poorly defined residues 31 to 34. Multiple conformations are observed for four cysteine residues and an MPD oxygen atom. The electron density indicates that disulfide bonds between Cys12 and Cys65 and between Cys29 and Cys48 have two distinct side-chain conformations. A molecule of MPD bridges neighboring protein molecules in the crystal lattice, and both MPD enantiomers are present in the crystal. A total of 125 water molecules per molecule of protein are included in the final model with B-values ranging from 11 to 52 A2 and occupancies from unity down to 0.4. Comparisons between the 1.2 A and 1.8 A models, including the bound water structure and crystal packing contacts, are emphasized.     

Validation of NMR side-chain conformations by packing calculations

The precision and accuracy of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy depend on the completeness of input experimental data set. Typically, rather than a single structure, an ensemble of up to 20 equally representative conformers is generated and routinely deposited in the Protein Database. There are substantially more experimentally derived restraints available to define the main-chain coordinates than those of the side chains. Consequently, the side-chain conformations among the conformers are more variable and less well defined than those of the backbone. Even when a side chain is determined with high precision and is found to adopt very similar orientations among all the conformers in the ensemble, it is possible that its orientation might still be incorrect. Thus, it would be helpful if there were a method to assess independently the side-chain orientations determined by NMR. Recently, homology modeling by side-chain packing algorithms has been shown to be successful in predicting the side-chain conformations of the buried residues for a protein when the main-chain coordinates and sequence information are given. Since the main-chain coordinates determined by NMR are consistently more reliable than those of the side-chains, we have applied the side-chain packing algorithms to predict side-chain conformations that are compatible with the NMR-derived backbone. Using four test cases where the NMR solution structures and the X-ray crystal structure of the same protein are available, we demonstrate that the side-chain packing method can provide independent validation for the side-chain conformations of NMR structures. Comparison of the side-chain conformations derived by side-chain packing prediction and by NMR spectroscopy demonstrates that when there is agreement between the NMR model and the predicted model, on average 78% of the time the X-ray structure also concurs. While the side-chain packing method can confirm the reliable residue conformations in NMR models, more importantly, it can also identify the questionable residue conformations with an accuracy of 60%. This validation method can serve to increase the confidence level for potential users of structural models determined by NMR.     

Conformational properties of trans Ac-Asn-Pro-Tyr-NHMe and trans Ac-Tyr-Pro-Asn-NHMe in dimethylsulfoxide and in water determined by multinuclear n.m.r. spectroscopy

Vicinal coupling constants between various nuclei provide backbone and side-chain conformational information for a series of asparagine- and tyrosine-containing peptides in DMSO and in H2O. By enriching Tyr of Ac-Asn-Pro-Tyr-NHMe with 15N, it has been possible to distinguish between the resonances of the two side-chain beta protons of Tyr. Analysis of the coupling constants in terms of the distributions of side-chain conformations in these peptides indicates that the addition of Asn to the Pro-Tyr sequence leads to a less random conformational distribution. When compared to the side-chain rotamer distribution of Ac-Asn-NHMe and Ac-Tyr-NHMe, particular Asn and Tyr side-chain conformations of Ac-Asn-Pro-Tyr-NHMe are stabilized in dimethylsulfoxide solution. The interaction(s) which stabilize a unique Tyr side-chain conformation of Ac-Asn-Pro-Tyr-NHMe in dimethylsulfoxide are not present in Ac-Ala-Pro-Tyr-NHMe and are unaffected by the addition of Val-Pro to the C-terminus of Asn-Pro-Tyr. In water, a preferential stabilization of one Asn side-chain conformation of Ac-Asn-Pro-Tyr-NHMe is also observed, while the Tyr side-chain rotamer distribution is similar to that of Ac-Tyr-NHMe. An interaction between the Asn side chain and the Pro-Tyr-NHMe backbone was previously shown to stabilize a beta-bend conformation at Pro-Tyr in water. Data are also presented for Ac-Tyr-Pro-Asn-NHMe, for which local interactions do not stabilize particular backbone conformations in dimethylsulfoxide or in water. The conformations of the peptides studied here are relatively insensitive to temperatures between 27 degrees and 62 degrees, both in dimethylsulfoxide and in water. The sequences Asn-Pro-Tyr and Tyr-Pro-Asn occur in ribonuclease A, and these tripeptides serve as models for the interactions involved in the folding of this protein.     

On residues in the disallowed region of the Ramachandran map.

An analysis of the occurrence of nonglycyl residues in conformations disallowed in the Ramachandran plot is presented. Ser, Asn, Thr, and Cys have the highest propensities to exhibit such conformations, and the branched aliphatic residues the lowest. Residues cluster in five regions and there are some trends in the types of residues and their side-chain conformations (chi(1)) occupying these. Majority of the residues are found at the edge of helices and strands and in short loops, and are involved in different types of weak, stabilizing interactions. A structural motif has been identified where a residue in disallowed conformation occurs as the first residue of a short 3(10)-helix. On the basis of the types of neighboring residues, the location in the three-dimensional structure and accessibility, there are similarities with the occurrence of cis peptide bonds in protein structures.     

Conformation of di-n-propylglycine residues (Dpg) in peptides: crystal structures of a type I' beta-turn forming tetrapeptide and an alpha-helical tetradecapeptide.

The crystal structures of two oligopeptides containing di-n-propylglycine (Dpg) residues, Boc-Gly-Dpg-Gly-Leu-OMe (1) and Boc-Val-Ala-Leu-Dpg-Val-Ala-Leu-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe (2) are presented. Peptide 1 adopts a type I'beta-turn conformation with Dpg(2)-Gly(3) at the corner positions. The 14-residue peptide 2 crystallizes with two molecules in the asymmetric unit, both of which adopt alpha-helical conformations stabilized by 11 successive 5 --> 1 hydrogen bonds. In addition, a single 4 --> 1 hydrogen bond is also observed at the N-terminus. All five Dpg residues adopt backbone torsion angles (phi, psi) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-C(alpha)-C' (tau) and the observed backbone phi,psi values. For tau > 106 degrees, helices are observed, while fully extended structures are characterized by tau < 106 degrees. The mean tau values for extended and folded conformations for the Dpg residue are 103.6 degrees +/- 1.7 degrees and 109.9 degrees +/- 2.6 degrees, respectively.     

Analysis of side-chain rotamers in transmembrane proteins

We measured the frequency of side-chain rotamers in 14 alpha-helical and 16 beta-barrel membrane protein structures and found that the membrane environment considerably perturbs the rotamer frequencies compared to soluble proteins. Although there are limited experimental data, we found statistically significant changes in rotamer preferences depending on the residue environment. Rotamer distributions were influenced by whether the residues were lipid or protein facing, and whether the residues were found near the N- or C-terminus. Hydrogen-bonding interactions with the helical backbone perturbs the rotamer populations of Ser and His. Trp and Tyr favor side-chain conformations that allow their side chains to extend their polar atoms out of the membrane core, thereby aligning the side-chain polarity gradient with the polarity gradient of the membrane. Our results demonstrate how the membrane environment influences protein structures, providing information that will be useful in the structure prediction and design of transmembrane proteins.     

Some observations on conserved polar side chains in immunoglobulin V-domains

1. The roles of conserved polar residues have been studied in 12 V-domains for which atomic coordinates are available. 2. In most cases a particular residue had a similar side chain conformation in all V-domains examined and the polar group provided the same hydrogen bonds which helped to stabilize the conformations of the domains. 3. In the case of a conserved glutamine/glutamic acid residue the buried side chain could adopt a variety of conformations and the polar group could form different hydrogen bonds from one domain to another. However, they contributed similarly to domain stability. 4. In the case of a conserved threonine/serine residue its side chain showed relative rotations of up to 180 degrees from one domain to another. The hydroxyl group could be buried or exposed at the domain surface. In some domains it formed hydrogen bonds to two other protein atoms but in other domains there was a single hydrogen bond or none at all. The varied roles of this residue are discussed in the text.     

Receptor rigidity and ligand mobility in trypsin-ligand complexes

The trypsin-like serine proteases comprise a structurally similar family of proteins with a wide diversity of biological functions. Members of this family play roles in digestion, hemostasis, immune responses, and cancer metastasis. Bovine trypsin is an archetypical member of this family that has been extensively characterized both functionally and structurally, and that preferentially hydrolyzes Arg/Lys-Xaa peptide bonds. We have used molecular dynamics (MD) simulations to study bovine trypsin complexed with the two noncovalent small-molecule ligands, benzamidine and tranylcypromine, that have the same hydrogen-bond donating moieties as Arg and Lys side-chains, respectively. Multiple (10) simulations ranging from 1 ns to 2.2 ns, with explicit water molecules and periodic boundary conditions, were performed. The simulations reveal that the trypsin binding pocket residues are relatively rigid regardless of whether there is no ligand, a high-affinity ligand (benzamidine), or a low-affinity ligand (tranylcypromine). The thermal average of the conformations sampled by benzamidine bound to trypsin is planar and consistent with the planar internal geometry of the benzamidine crystallographic model coordinates. However, the most probable bound benzamidine conformations are +/-25 degrees out of plane, implying that the observed X-ray electron density represents an average of densities from two mirror symmetric, nonplanar conformations. Solvated benzamidine has free energy minima at +/-45 degrees , and the induction of a more planar geometry upon binding is associated with approximately 1 kcal/mol of intramolecular strain. Tranylcypromine's hydrogen-bonding pattern in the MD differs substantially from that inferred from the X-ray electron density. Early in simulations of this system, tranylcypromine adopts an alternative binding conformation, changing from the crystallographic conformation, with a direct hydrogen bond between its amino moiety and the backbone oxygen of Gly219, to one having a bridging water molecule. This result is consistently seen with the CHARMM22, Amber, or OPLS-AA force fields. The trypsin-tranylcypromine hydrogen-bonding pattern observed in the simulations also occurs as the crystallographic binding mode of the Lys15 side-chain of bovine pancreatic trypsin inhibitor bound to trypsin. In this latter cocrystal, a bridging crystallographic water does reside between the side-chain's amino group and the trypsin Gly219 backbone oxygen. Furthermore, the trypsin-tranylcypromine simulations sample two different stable noncrystallographic binding poses. These data suggest that some of the electron density ascribed to tranylcypromine in the X-ray model is rather due to a bound water molecule, and that multiple tranylcypromine binding conformations (crystallographic disorder) may be the cause of ambiguous electron density. The combined trypsin-benzamidine and trypsin- tranylcypromine results highlight the ability of simulations to augment protein-ligand complex structural data by deconvoluting the effects of thermal and structural averaging, and by finding energetically optimal ligand and bound water positions for weakly bound ligands.