Csaba Magyar

SRide: a server for identifying stabilizing residues in proteins

Residues expected to play key roles in the stabilization of proteins [stabilizing residues (SRs)] are selected by combining several methods based mainly on the interactions of a given residue with its spatial, rather than its sequential neighborhood and by considering the evolutionary conservation of the residues. A residue is selected as a stabilizing residue if it has high surrounding hydrophobicity, high long-range order, high conservation score and if it belongs to a stabilization center. The definition of all these parameters and the thresholds used to identify the SRs are discussed in detail. The algorithm for identifying SRs was originally developed for TIM-barrel proteins [M. M. Gromiha, G. Pujadas, C. Magyar, S. Selvaraj, and I. Simon (2004), Proteins, 55, 316–329] and is now generalized for all proteins of known 3D structure. SRs could be applied in protein engineering and homology modeling and could also help to explain certain folds with significant stability. The SRide server is located at .

Locating the stabilizing residues in (α/β)8 barrel proteins based on hydrophobicity, long‐range interactions, and sequence conservation

In nature, 1 out of every 10 proteins has an (α/β)8 (TIM)‐barrel fold, and in most cases, pairwise comparisons show no sequence similarity between them. Hence, delineating the key residues that induce very different sequences to share a common fold is important for understanding the folding and stability of TIM‐barrel domains. In this work, we propose a new consensus approach for locating these stabilizing residues based on long‐range interactions, hydrophobicity, and conservation of amino acid residues. We have identified 957 stabilizing residues in 63 proteins from a nonredundant set of 71 TIM‐barrel domains. Most of these residues are located in the 8‐stranded β‐sheet, with nearly one half of them oriented toward the interior of the barrel and the other half oriented toward the surrounding α‐helices. Several stabilizing residues are found in the N‐ and C‐terminal loops, whereas very few appear in the α‐helices that surround the internal β‐sheet. Further, these 957 residues are placed in 434 stabilizing segments of various sizes, and each domain contains 1–10 of these segments. We found that 8 segments per domain is the most abundant one, and two thirds of the proteins have 7–9 stabilizing segments. Finally, we verified the identified residues with experimental temperature factors and found that these residues are among the ones with less mobility in the considered proteins. We suggest that our new protocol serves as a powerful tool to identify the stabilizing residues in TIM‐barrel domains, which can be used as potential candidates for studying protein folding and stability by means of protein engineering experiments. Proteins 2004;55:000–000.

Flexibility of prolyl oligopeptidase : Molecular dynamics and molecular framework analysis of the potential substrate pathways

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven‐bladed β‐propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter‐domain region comprising the highly flexible N‐terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192–205) was identified by cross‐correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains. Proteins 2005.

Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

Phosphorylation-induced transient intrinsic structure in the kinase-inducible domain of CREB facilitates its recognition by the KIX domain of CBP

Phosphorylation at Ser‐133 of the kinase inducible domain of CREB (KID) triggers its binding to the KIX domain of CBP via a concomitant coil‐to‐helix transition. The exact role of this key event is still puzzling: it does not switch between disordered and ordered states, nor its direct interactions fully account for selectivity. Hence, we reasoned that phosphorylation may shift the conformational preferences of KID towards a binding‐competent state. To this end we investigated the intrinsic structural properties of the unbound KID in phosphorylated and unphosphorylated forms by simulated annealing and molecular dynamics simulations. Although helical populations show subtle differences, phosphorylation reduces the flexibility of the turn segment connecting the two helices in the complexed structure and induces a transient structural element that corresponds to its bound conformation. It is stabilized by the pSer‐133–Arg‐131 interaction, which is absent from the unphosphorylated KID. Diminishing this coupling decreases the 3.1 kcal/mol contribution of pSer‐133 to the binding free energy (ΔGbind) of the phosphorylated KID to KIX by 1.1 kcal/mol, as computed in reference to Ser‐133. In a binding competent form of the S133E KID mutant, the contribution of Glu‐133 to ΔGbind is by 1.5 kcal/mol smaller than that of pSer, suggesting that altered structural properties due to pSer → Glu replacement impair the binding affinity. Thus, we propose that phoshorylation contributes to selectivity not merely by the direct interactions of the phosphate group with KIX, but also by promoting the formation of a transient structural element in the highly conserved turn segment. Proteins 2006.

Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3-isopropylmalate dehydrogenase

The comparison of the three‐dimensional structures of thermophilic (Thermus thermophilus) and mesophilic (Escherichia coli) 3‐isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability. As a test of our assumption, glutamine 200 in the E. coli enzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters. At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair. These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.

Combination of 2D/3D Ligand-Based Similarity Search in Rapid Virtual Screening from Multimillion Compound Repositories. Selection and Biological Evaluation of Potential PDE4 and PDE5 Inhibitors

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost effective approach. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compound databases but the generated library requires further focusing by various 2D/3D chemoinformatics tools. We report here a combination of the 2D approach with a ligand-based 3D method (Screen3D) which applies flexible matching to align reference and target compounds in a dynamic manner and thus to assess their structural and conformational similarity. In the first case study we compared the 2D and 3D similarity scores on an existing dataset derived from the biological evaluation of a PDE5 focused library. Based on the obtained similarity metrices a fusion score was proposed. The fusion score was applied to refine the 2D similarity search in a second case study where we aimed at selecting and evaluating a PDE4B focused library. The application of this fused 2D/3D similarity measure led to an increase of the hit rate from 8.5% (1st round, 47% inhibition at 10 µM) to 28.5% (2nd round at 50% inhibition at 10 µM) and the best two hits had 53 nM inhibitory activities.

Functionally and structurally relevant residues of enzymes: are they segregated or overlapping?

There is a delicate balance between stability and flexibility needed for enzyme function. To avoid undesirable alteration of the functional properties during the evolutionary optimization of the structural stability under certain circumstances, and vice versa, to avoid unwanted changes of stability during the optimization of the functional properties of proteins, common sense would suggest that parts of the protein structure responsible for stability and parts responsible for function developed and evolved separately. This study shows that nature did not follow this anthropomorphic logic: the set of residues involved in function and those involved in structural stabilization of enzymes are rather overlapping than segregated.

Noncovalent cross-links in context with other structural and functional elements of proteins.

Proteins are heteropolymers with evolutionary selected native sequences of residues. These native sequences code for unique and stable 3D structures indispensable for biochemical activity and for proteolysis resistance, the latter which guarantees an appropriate lifetime for the protein in the protease rich cellular environment. Cross-links between residues close in space but far in the primary structure are required to maintain the folded structure of proteins. Some of these cross-links are covalent, most frequently disulfide bonds, but the majority of the cross-links are sets of cooperative noncovalent long-range interactions. In this paper we focus on special clusters of noncovalent long-range interactions: the Stabilization Centers (SCs). The relation between the SCs and secondary structural elements as well as the relation between SCs and functionally important regions of proteins are presented to show a detailed picture of these clusters, which are believed to be primarily responsible for major aspects of protein stability.

Combination of Pharmacophore Matching, 2D Similarity Search, and In Vitro Biological Assays in the Selection of Potential 5-HT6 Antagonists from Large Commercial Repositories

Rapid in silico selection of target‐focused libraries from commercial repositories is an attractive and cost‐effective approach. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compound databases, but the generated library requires further focusing. We report here a combination of the 2D approach with pharmacophore matching which was used for selecting 5‐HT6 antagonists. In the first screening round, 12 compounds showed >85% antagonist efficacy of the 91 screened. For the second‐round (hit validation) screening phase, pharmacophore models were built, applied, and compared with the routine 2D similarity search. Three pharmacophore models were created based on the structure of the reference compounds and the first‐round hit compounds. The pharmacophore search resulted in a high hit rate (40%) and led to novel chemotypes, while 2D similarity search had slightly better hit rate (51%), but lacking the novelty. To demonstrate the power of the virtual screening cascade, ligand efficiency indices were also calculated and their steady improvement was confirmed.