Side-chain Repacking Research Articles

Volume-based solvation models out-perform area-based models in combined studies of wild-type and mutated protein-protein interfaces

BackgroundEmpirical binding models have previously been investigated for the energetics of protein complexation (ΔG models) and for the influence of mutations on complexation (i.e. differences between wild-type and mutant complexes, ΔΔG models). We construct binding models to directly compare these processes, which have generally been studied separately.ResultsAlthough reasonable fit models were found for both ΔG and ΔΔG cases, they differ substantially. In a dataset curated for the absence of mainchain rearrangement upon binding, non-polar area burial is a major determinant of ΔG models. However this ΔG model does not fit well to the data for binding differences upon mutation. Burial of non-polar area is weighted down in fitting of ΔΔG models. These calculations were made with no repacking of sidechains upon complexation, and only minimal packing upon mutation. We investigated the consequences of more extensive packing changes with a modified mean-field packing scheme. Rather than emphasising solvent exposure with relatively extended sidechains, rotamers are selected that exhibit maximal packing with protein. This provides solvent accessible areas for proteins that are much closer to those of experimental structures than the more extended sidechain regime. The new packing scheme increases changes in non-polar burial for mutants compared to wild-type proteins, but does not substantially improve agreement between ΔG and ΔΔG binding models.ConclusionWe conclude that solvent accessible area, based on modelled mutant structures, is a poor correlate for ΔΔG upon mutation. A simple volume-based, rather than solvent accessibility-based, model is constructed for ΔG and ΔΔG systems. This shows a more consistent behaviour. We discuss the efficacy of volume, as opposed to area, approaches to describe the energetic consequences of mutations at interfaces. This knowledge can be used to develop simple computational screens for binding in comparative modelled interfaces.

A knowledge‐based forcefield for protein–protein interface design

A distance-dependent knowledge-based potential for protein-protein interactions is derived and tested for application in protein design. Information on residue type specific C(alpha) and C(beta) pair distances is extracted from complex crystal structures in the Protein Data Bank and used in the form of radial distribution functions. The use of only backbone and C(beta) position information allows generation of relative protein-protein orientation poses with minimal sidechain information. Further coarse-graining can be done simply in the same theoretical framework to give potentials for residues of known type interacting with unknown type, as in a one-sided interface design problem. Both interface design via pose generation followed by sidechain repacking and localized protein-protein docking tests are performed on 39 nonredundant antibody-antigen complexes for which crystal structures are available. As reference, Lennard-Jones potentials, unspecific for residue type and biasing toward varying degrees of residue pair separation are used as controls. For interface design, the knowledge-based potentials give the best combination of consistently designable poses, low RMSD to the known structure, and more tightly bound interfaces with no added computational cost. 77% of the poses could be designed to give complexes with negative free energies of binding. Generally, larger interface separation promotes designability, but weakens the binding of the resulting designs. A localized docking test shows that the knowledge-based nature of the potentials improves performance and compares respectably with more sophisticated all-atoms potentials.

Automated computation of low‐energy pathways for complex rearrangements in proteins: Application to the conformational switch of Ras p21

The computation of minimum energy paths (MEPs) is an approach for gaining insight into protein conformational transitions that are too slow to be observed with unconstrained molecular dynamics simulations. MEPs have the advantage of providing the energy barrier of the rate-limiting step(s), allowing discrimination among different paths. Finding low-energy MEPs for complex transitions, such as those involving rearrangements of the backbone fold or repacking of buried side chains, has hitherto been unfeasible in a reliable, automated manner, the MEP often displaying unphysical behavior, such as the crossing of bonds. Here, this problem is addressed by combining a counterintuitive procedure for generating an initial guess of the path, in which all side chains are shrunk, with the conjugate peak refinement (CPR) method. The effectiveness of the approach is tested on the conformational switch in Ras p21. This conformational transition involves some partial unfolding and re-folding, a process for which a multitude of pathways are likely to exist and for which a single MEP does not provide a complete description. However, this transition requires some sterically demanding rearrangements, thus testing the ability of a method to find low-energy pathways free of structurally unphysical events. This is achieved by the present approach, which finds a path whose rate-limiting barrier is compatible with experiment. This demonstrates that the method can be used to compute plausible pathways for complex rearrangements in proteins in an automated manner that is unbiased by external driving constraints.

From synthetic coiled coils to functional proteins: automated design of a receptor for the calmodulin-binding domain of calcineurin

A series of synthetic receptors capable of binding to the calmodulin-binding domain of calcineurin (CN 393–414) was designed, synthesized and characterized. The design was accomplished by docking CN 393–414 against a two-helix receptor, using an idealized three-stranded coiled coil as a starting geometry. The sequence of the receptor was chosen using a side-chain re-packing program, which employed a genetic algorithm to select potential binders from a total of 7.5 × 10 6 possible sequences. A total of 25 receptors were prepared, representing 13 sequences predicted by the algorithm as well as 12 related sequences that were not predicted. The receptors were characterized by CD spectroscopy, analytical ultracentrifugation, and binding assays. The receptors predicted by the algorithm bound CN 393–414 with apparent dissociation constants ranging from 0.2 μM to >50 μM. Many of the receptors that were not predicted by the algorithm also bound to CN 393–414. Methods to circumvent this problem and to improve the automated design of functional proteins are discussed.

Derivation and testing of pair potentials for protein folding. When is the quasichemical approximation correct?

Many existing derivations of knowledge-based statistical pair potentials invoke the quasichemical approximation to estimate the expected side-chain contact frequency if there were no amino acid pair-specific interactions. At first glance, the quasichemical approximation that treats the residues in a protein as being disconnected and expresses the side-chain contact probability as being proportional to the product of the mole fractions of the pair of residues would appear to be rather severe. To investigate the validity of this approximation, we introduce two new reference states in which no specific pair interactions between amino acids are allowed, but in which the connectivity of the protein chain is retained. The first estimates the expected number of side-chain contracts by treating the protein as a Gaussian random coil polymer. The second, more realistic reference state includes the effects of chain connectivity, secondary structure, and chain compactness by estimating the expected side-chain contrast probability by placing the sequence of interest in each member of a library of structures of comparable compactness to the native conformation. The side-chain contact maps are not allowed to readjust to the sequence of interest, i.e., the side chains cannot repack. This situation would hold rigorously if all amino acids were the same size. Both reference states effectively permit the factorization of the side-chain contact probability into sequence-dependent and structure-dependent terms. Then, because the sequence distribution of amino acids in proteins is random, the quasichemical approximation to each of these reference states is shown to be excellent. Thus, the range of validity of the quasichemical approximation is determined by the magnitude of the side-chain repacking term, which is, at present, unknown. Finally, the performance of these two sets of pair interaction potentials as well as side-chain contact fraction-based interaction scales is assessed by inverse folding tests both without and with allowing for gaps.

Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme.

High resolution crystal structures have been determined for six chicken-type lysozymes that were constructed to investigate putative intermediates in the evolution of the lysozymes of modern game birds (Malcolm, B. A., Wilson, K. P., Matthews, B. W., Kirsch, J. F., and Wilson, A. C. (1990) Nature 345, 86-89). The amino acid replacements include Thr-40—-Ser, Ile-55—-Val, and Ser-91—-Thr, as well as combinations of these substitutions. Residues 40, 55, and 91 are buried within the core of chicken lysozyme. The replacements therefore involve the insertion and/or removal of methyl groups from the protein interior. The mutant proteins have normal activities, and their thermal stabilities span a range of 7 degrees C, with some variants more stable and some less stable than the naturally occurring forms. Comparison of the crystal structures shows the overall structures to be very similar, but there are differences in the packing of side chains in the region of the replacements. The x-ray coordinates were used to evaluate the repacking of side chains in the protein interior and to attempt to evaluate the contributions of the different energetic interactions toward the overall stability of each variant. The results illustrate how proteins can compensate for potentially destabilizing substitutions in different ways and underscore the importance of high resolution structural data if changes in protein thermostability due to changes in protein sequence are to be understood. The findings also suggest that protein stability can be increased by mutations that lower strain in the protein interior while maintaining total buried hydrophobic surface area.

Analysis of protein loop closure

As shown in previous crystallographic investigations, upon binding lactate and NAD, lactate dehydrogenase undergoes a large conformational change that results in a surface loop moving roughly 10 A to cover the active site. In addition, there are appreciable movements (approximately 2 A) of five helices and three other loops. We demonstrate by a new fitting procedure that the loop moves on two hinges separated by a relatively rigid type II turn. The first hinge has few steric constraints on it, and its motion can be well accounted for by large changes in two torsion angles, i.e. as in a classic hinge motion. In contrast, the second hinge, which is part of a helix connected to the end of the loop, has many more constraints on it and distributes its deformation over more torsion angles. This novel motion involves the helix stretching and splitting into alpha-helical and 3(10)-helical components and substantial side-chain repacking in the sense of "cogs hopping between grooves" at its interface with the end of a neighboring helix. The loop is stabilized by five transverse (across loop) hydrogen bonds. These are preserved, through the conformational change and through 17 lactate dehydrogenase sequences, more than the longitudinal hydrogen bonds down the sides of the loop. Through a network of contacts, many of them conserved hydrophobic residues, the motion of the loop is propagated outward to structures that have no direct contact with the ligands. These moving structures are on the surface of the protein, and the whole protein can be subdivided into concentric shells of increasing mobility.

Toward a simplification of the protein folding problem: a stabilizing polyalanine .alpha.-helix engineered in T4 lysozyme

In an attempt to simplify the protein folding problem, and also to further investigate the role of alanine as a helix-stabilizing residue, a series of alanines was introduced within the alpha-helix that includes residues 126-134 of T4 lysozyme. In wild-type lysozyme this alpha-helix contains alanine residues at positions 129, 130, and 134. Mutant lysozymes with alanines substituted at positions 128, 131, 132, and 133, either as single substitutions or in selected combinations, were constructed by oligonucleotide-directed mutagenesis. With the exception of the replacement of Leu 133, which is buried within the hydrophobic core of the protein, all the variants were more stable than wild-type lysozyme. The variant with alanines substituted at positions 128, 131, and 132 (E128A/V131A/N132A), which incorporates the sequence Ala 128-Ala 129-Ala 130-Ala 131-Ala 132-Leu 133-Ala 134, has a melting temperature 3.3 degrees C above that of wild-type lysozyme. Determination of the crystal structure of this mutant lysozyme shows that the replacement of Glu 128, Val 131, and Asn 132 with alanine causes alpha-helix 126-134 to rotate 3.4 degrees about an axis parallel to its own axis. This rotation seems to be triggered primarily by the loss of a hydrogen bond between Asn 132 and Ser 117 and is associated with the repacking of several side chains at the interface between alpha-helix 126-134 and the adjacent alpha-helix 115-122.(ABSTRACT TRUNCATED AT 250 WORDS)