The role of carbon-donor hydrogen bonds in stabilizing tryptophan conformations

Although most side-chain torsion angles correspond to low-energy rotameric positions, deviations occur with significant frequency. One striking example arises in Trp residues, which have an important role in stabilizing protein structures because of their size and mixed hydrophobic/hydrophilic character. Ten percent of Trp side-chains have unexplained conformations with chi(2) near 0 degrees instead of the expected 90 degrees. The current work is a structural and energetic analysis of these conformations. It is shown that many Trp residues with these orientations are stabilized by three-center carbon-donor hydrogen bonds of the form C-H...X...H-C, where X is a polar hydrogen-bond acceptor in the environment of the side-chain. The bridging hydrogen bonds occur both within the Trp side-chain and between the side-chain and the local protein backbone. Free energy maps of an isolated Trp residue in an explicit water environment show a minimum corresponding to the off-rotamer peak observed in the crystallographic data. Bridging carbon-donor hydrogen bonds are also shown to stabilize on-rotamer Trp conformations, and similar bridging hydrogen bonds also stabilize some off-rotamer Asp conformations. The present results suggest a previously unrecognized role for three-center carbon-donor hydrogen bonds in protein structures and support the view that the off-rotamer Trp side-chain orientations are real rather than artifacts of crystallographic refinements. Certain of the off-rotamer Trp conformations appear to have a functional role.     

Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain

The first step in both normal haemostasis and arterial thrombosis is the interaction between collagen, von Willebrand factor (vWF), and glycoprotein Ib. The A3 domain of vWF forms the principal binding site for collagen type I and type III. Inhibition of the vWF-collagen interaction by an anti-human vWF monoclonal antibody (MoAb) 82D6A3 can be a potential way to prevent arterial thrombosis. Identification of the epitope of MoAb 82D6A3 showed recently that the consensus sequence SPWR obtained by phage display could adopt the conformation of the discontinuous epitope. Modelling showed that Trp982 in the vWF had to obtain a more solvent accessible conformation. We performed a detailed fluorescence study of Trp982 in the vWF A3. Using the method described by Hellings et al. (Biophys J 2003;85:1894-1902), we were able to identify two different low-energy Trp982 rotamers and to link them with their experimentally derived fluorescence lifetimes. Fluorescence anisotropy showed no interconversion in the nanosecond timescale between the two different rotameric states. With these experiments, we gather strong indications for the existence of an exposed rotamer conformation and a rotamer that corresponds to the one observed in the X-ray structure. These results strongly support the modeling work (Vanhoorelbeke et al., J Biol Chem 2003;278:37815-37821).     

Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease

The Q7K/L331/L631 HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with k(q1) = 6.85 x 10(9) M(-1) s(-1) and k(q2) = 1.88 x 10(9) M(-1) s(-1), that merge into one in the complex with k(q) = 1.78 x 10(9) M(-1) s(-1). 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.     

A test of enhancing model accuracy in high-throughput crystallography

The high throughput of structure determination pipelines relies on increased automation and, consequently, a reduction of time spent on interactive quality control. In order to meet and exceed current standards in model accuracy, new approaches are needed for the facile identification and correction of model errors during refinement. One such approach is provided by the validation and structure-improvement tools of the MOLPROBITY web service. To test their effectiveness in high-throughput mode, a large subset of the crystal structures from the SouthEast Collaboratory for Structural Genomics (SECSG) has used protocols based on the MOLPROBITY tools. Comparison of 29 working-set and 19 control-set SECSG structures shows that working-set outlier scores for updated Ramachandran-plot, sidechain rotamer, and all-atom steric criteria have been improved by factors of 5- to 10-fold (relative to the control set or to a Protein Data Bank sample), while quality of covalent geometry, R_work, R_free, electron density and difference density are maintained or improved. Some parts of this correction process are already fully automated; other parts involve manual rebuilding of conformations flagged by the tests as trapped in the wrong local minimum, often altering features of functional significance. The ease and effectiveness of this technique shows that macromolecular crystal structures from either traditional or high-throughput determinations can feasibly reach a new level of excellence in conformational accuracy and reliability.     

Modeling the anti-CEA antibody combining site by homology and conformational search.

A model for an antibody specific for the carcinoembryonic antigen (CEA) has been constructed using a method which combines the concept of canonical structures with conformational search. A conformational search technique is introduced which couples random generation of backbone loop conformations to a simulated annealing method for assigning side chain conformations. This technique was used both to verify conformations selected from the set of known canonical structures and to explore conformations available to the H3 loop in CEA ab initio. Canonical structures are not available for H3 due to its variability in length, sequence, and observed conformation in known antibody structures. Analysis of the results of conformational search resulted in three equally probable conformations for H3 loop in CEA. Force field energies, solvation free energies, exposure of charged residues and burial of hydrophobic residues, and packing of hydrophobic residues at the base of the loop were used as selection criteria. The existence of three equally plausible structures may reflect the high degree of flexibility expected for an exposed loop of this length. The nature of the combining site and features which could be important to interaction with antigen are discussed.     

Current developments in Coot for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data

Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo‐EM model building and refinement, as cryo‐EM structures with resolution ranging 2.5–4 A are now routinely available. Model building into these maps can be time‐consuming and requires experience in both biochemistry and building into low‐resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman‐McClure restraints, full‐chain refinement, and Fourier‐model based residue‐type‐specific Ramachandran restraints. Here, we present the current state‐of‐the‐art in Coot usage.     

The use of position-specific rotamers in model building by homology

In this study we concentrate on replacing side chains as a subtask of model building by homology. Two problems arise. How to determine potential low energy rotamers? And how to avoid the combinatorial explosion that results from the combination of many residues for which multiple good rotamers are predicted? We attempt to solve these problems by choosing position-specific rather than generalized rotamers and by sorting the residues that have to be modelled as a function of their freedom in rotamer space. The practical advantages of our method are the quality of the models for cases of high backbone similarity, the small amount of human intervention needed, and the fact that the method automatically estimates the reliability with which each residue has been modeled. Other methods described in this issue are probably more suitable if large backbone rearrangements or loop insertions and deletions need to be modeled. © 1995 Wiley-Liss, Inc.     

Isolation and identification of flavonoids, including flavone rotamers, from the herbal drug 'Crataegi folium cum flore' (hawthorn)

Twelve flavonoids, including seven flavones, four flavonols and one flavanone, were isolated from methanolic extract of the herbal drug 'Crataegi folium cum flore' (hawthorn leaves and flowers) by a combination of CC (over Amberlite XAD-7 and Sephadex LH-20) and preparative HPLC. Their structures, including that of the novel flavonol 8-methoxykaempferol 3-O-(6"-malonyl-beta-glucopyranoside), were elucidated by homo- and heteronuclear NMR and electrospray/MS. The 1H- and 13C-NMR of all compounds, including rotameric pairs of five flavone C-glycosides, were assigned. The presence and relative proportion of each rotamer was shown by various NMR experiments, including two-dimensional nuclear Overhauser and exchange spectroscopy, to depend on solvent, linkage position and structure of the C-glycosyl substituent.     

Fast and simple Monte Carlo algorithm for side chain optimization in proteins: application to model building by homology.

An unknown protein structure can be predicted with fair accuracy once an evolutionary connection at the sequence level has been made to a protein of known 3-D structure. In model building by homology, one typically starts with a backbone framework, rebuilds new loop regions, and replaces nonconserved side chains. Here, we use an extremely efficient Monte Carlo algorithm in rotamer space with simulated annealing and simple potential energy functions to optimize the packing of side chains on given backbone models. Optimized models are generated within minutes on a workstation, with reasonable accuracy (average of 81% side chain chi 1 dihedral angles correct in the cores of proteins determined at better than 2.5 A resolution). As expected, the quality of the models decreases with decreasing accuracy of backbone coordinates. If the back-bone was taken from a homologous rather than the same protein, about 70% side chain chi 1 angles were modeled correctly in the core in a case of strong homology and about 60% in a case of medium homology. The algorithm can be used in automated, fast, and reproducible model building by homology.     

Side-chain conformational changes upon Protein-Protein Association

Conformational changes upon protein-protein association are the key element of the binding mechanism. The study presents a systematic large-scale analysis of such conformational changes in the side chains. The results indicate that short and long side chains have different propensities for the conformational changes. Long side chains with three or more dihedral angles are often subject to large conformational transition. Shorter residues with one or two dihedral angles typically undergo local conformational changes not leading to a conformational transition. A relationship between the local readjustments and the equilibrium fluctuations of a side chain around its unbound conformation is suggested. Most of the side chains undergo larger changes in the dihedral angle most distant from the backbone. The frequencies of the core-to-surface interface transitions of six nonpolar residues and Tyr are larger than the frequencies of the opposite surface-to-core transitions. The binding increases both polar and nonpolar interface areas. However, the increase of the nonpolar area is larger for all considered classes of protein complexes, suggesting that the protein association perturbs the unbound interfaces to increase the hydrophobic contribution to the binding free energy. To test modeling approaches to side-chain flexibility in protein docking, conformational changes in the X-ray set were compared with those in the docking decoy sets. The results lead to a better understanding of the conformational changes in proteins and suggest directions for efficient conformational sampling in docking protocols.