The limit of accuracy of protein modeling: influence of crystal packing on protein structure

The size of the protein database (PDB) makes it now feasible to arrive at statistical conclusions regarding structural effects of crystal packing. These effects are relevant for setting upper practical limits of accuracy on protein modeling. Proteins whose crystals have more than one molecule in the asymmetric unit or whose structures were determined at least twice by X-ray crystallography were paired and their differences analyzed. We demonstrate a clear influence of crystal environment on protein structure, including backbone conformations, hinge-like motions and side-chain conformations. The positions of surface water molecules tend to be variable in different crystal environments while those of ligands are not. Structures determined by independent groups vary more than structures determined by the same authors. The use of different refinement methods is a major source for this effect. Our pair-wise analysis derives a practical limit to the accuracy of protein modeling. For different crystal forms, the limit of accuracy (C(alpha), root-mean-square deviation (RMSD)) is approximately 0.8A for the entire protein, which includes approximately 0.3A due to crystal packing. For organized secondary elements, the upper limit of C(alpha) RMSD is 0.5-0.6A while for loops or protein surface it reaches 1.0A. Twenty percent of exposed side- chains exhibit different chi(1+2) conformations with approximately half of the effect also resulting from crystal packing. A web based tool for analysis and graphic presentation of surface areas of crystal contacts is available (http://ligin.weizmann.ac.il/cryco).     

Verification of protein structures: patterns of nonbonded atomic interactions.

A novel method for differentiating between correctly and incorrectly determined regions of protein structures based on characteristic atomic interaction is described. Different types of atoms are distributed nonrandomly with respect to each other in proteins. Errors in model building lead to more randomized distributions of the different atom types, which can be distinguished from correct distributions by statistical methods. Atoms are classified in one of three categories: carbon (C), nitrogen (N), and oxygen (O). This leads to six different combinations of pairwise noncovalently bonded interactions (CC, CN, CO, NN, NO, and OO). A quadratic error function is used to characterize the set of pairwise interactions from nine-residue sliding windows in a database of 96 reliable protein structures. Regions of candidate protein structures that are mistraced or misregistered can then be identified by analysis of the pattern of nonbonded interactions from each window.     

A dissection of specific and non-specific protein-protein interfaces

We compare the geometric and physical-chemical properties of interfaces involved in specific and non-specific protein-protein interactions in crystal structures reported in the Protein Data Bank. Specific interactions are illustrated by 70 protein-protein complexes and by subunit contacts in 122 homodimeric proteins; non-specific interactions are illustrated by 188 pairs of monomeric proteins making crystal-packing contacts selected to bury more than 800 A2 of protein surface. A majority of these pairs have 2-fold symmetry and form crystal dimers that cannot be distinguished from real dimers on the basis of the interface size or symmetry. The chemical and amino acid compositions of the large crystal-packing interfaces resemble the protein solvent-accessible surface. These interfaces are less hydrophobic than in homodimers and contain much fewer fully buried atoms. We develop a residue propensity score and a hydrophobic interaction score to assess preferences seen in the chemical and amino acid compositions of the different types of interfaces, and we derive indexes to evaluate the atomic packing, which we find to be less compact at non-specific than at specific interfaces. We test the capacity of these parameters to identify homodimeric proteins in crystal structures, and show that a simple combination of the non-polar interface area and the fraction of buried interface atoms assigns the quaternary structure of 88% of the homodimers and 77% of the monomers in our data set correctly. These success rates increase to 93-95% when the residue propensity score of the interfaces is taken into consideration.     

Primary and secondary interactions between CK2α and CK2β lead to ring-like structures in the crystals of the CK2 holoenzyme

Protein kinase CK2 predominantly exists as a heterotetrameric holoenyzme consisting of two catalytic subunits (CK2α) and two non-catalytic subunits (CK2β). Early investigations which we review here had revealed the presence of two types of contacts between CK2α and CK2β: a primary interaction responsible for the stability of the CK2 holoenzyme and stimulatory for the catalytic activity, and a secondary interaction which is inhibitory and in which the acidic loop of CK2β associates with the basic stretch and the (p+1)-loop of CK2α. At the end of the last decade both types of interactions were assumed to occur within the same tetrameric complex. The CK2 holoenyzme structure, however, suggested that the secondary interactions must happen between different CK2 tetramers. Such a behaviour should lead to higher-ordered aggregates consistent with several previous reports about a distinct aggregation propensity of CK2. We demonstrate here that in the CK2 holoenzyme crystals contacts between different CK2 tetramers exists which provide structural details of the secondary CK2α/CK2β interactions. These mainly ionic interactions lead to trimeric rings of CK2 holoenzymes in the crystal. In these rings each CK2 tetramer possesses one CK2α subunit open for substrate binding and another one whose active site is blocked by a secondary contact with CK2β from a neighbouring tetramer. This observation fits to previous findings that salt-sensitive ring-like aggregates of CK2 holoenzymes can exist which possess significant catalytic activity. Furthermore it suggests that earlier ideas about a regulatory role of the enzyme’s aggregation propensity may be worth to be revitalised.     

An empirical energy function for threading protein sequence through the folding motif

In this paper we present a new residue contact potantial derived by statistical analysis of protein crystal structures. This gives mean hydrophobic and pairwise contact energies as a function of residue type and distance interval. To test the accuracy of this potential we generate model structures by “threading” different sequences through backbone folding motifs found in the structural data base. We find that conformational energies calculated by summing contact potentials show perfect specificity in matching the correct sequences with each globular folding motif in a 161-protcin data set. They also identify correct models with the core folding motifs of heme-rythrin and immunoglobulin McPC603 V₁-do- main, among millions of alternatives possible when we align subsequences with α-helices and β-strands, and allow for variation in the lengths of intervening loops. We suggest that contact potentials reflect important constraints on nonbonded interaction in native proteins, and that “threading” may be useful for structure prediction by recognition of folding motif. © 1993 Wiley-Liss, Inc.     

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation. Here, we present the three-dimensional structure of the complex between a cytosolic domain of Pex3p and the binding-region peptide of Pex19p. The overall shape of Pex3p is a prolate spheroid with a novel fold, the 'twisted six-helix bundle.' The Pex19p-binding site is at an apex of the Pex3p spheroid. A 16-residue region of the Pex19p peptide forms an α-helix and makes a contact with Pex3p; this helix is disordered in the unbound state. The Pex19p peptide contains a characteristic motif, consisting of the leucine triad (Leu18, Leu21, Leu22), and Phe29, which are critical for the Pex3p binding and peroxisome biogenesis.     

Analysis of the quaternary structure of the MutL C-terminal domain

The dimeric DNA mismatch repair protein MutL has a key function in communicating mismatch recognition by MutS to downstream repair processes. Dimerization of MutL is mediated by the C-terminal domain, while activity of the protein is modulated by the ATP-dependent dimerization of the highly conserved N-terminal domain. Recently, a crystal structure analysis of the Escherichia coli MutL C-terminal dimerization domain has been reported and a model for the biological dimer was proposed. In this model, dimerization is mediated by the internal (In) subdomain comprising residues 475-569. Here, we report a computational analysis of all protein interfaces observed in the crystal structure and suggest that the biological dimer interface is formed by a hydrophobic surface patch of the external (Ex) subdomain (residues 432-474 and 570-615). Moreover, sequence analysis revealed that this surface patch is conserved among the MutL proteins. To test this hypothesis, single and double-cysteine variants of MutL were generated and tested for their ability to be cross-linked with chemical cross-linkers of various size. Finally, deletion of the C-terminal residues 605-615 abolished homodimerization. The biochemical data are fully compatible with a revised model for the biological dimer, which has important implications for understanding the heterodimerization of eukaryotic MutL homologues, modeling the MutL holoenzyme and predicting protein-protein interaction sites.