Biomedical subjects

Kannan Gunasekaran

Publications and source records attributed to Kannan Gunasekaran.

11 recordsLinked to original sources

PubMed2006

How different are structurally flexible and rigid binding sites? Sequence and structural features discriminating proteins that do and do not undergo conformational change upon ligand binding.

Proteins are dynamic molecules and often undergo conformational change upon ligand binding. It is widely accepted that flexible loop regions have a critical functional role in enzymes. Lack of consideration of binding site flexibility has led to failures in predicting protein functions and in successfully docking ligands with protein receptors. Here we address the question: which sequence and structural features distinguish the structurally flexible and rigid binding sites? We analyze high-resolution crystal structures of ligand bound (holo) and free (apo) forms of 41 proteins where no conformational change takes place upon ligand binding, 35 examples with moderate conformational change, and 22 cases where a large conformational change has been observed. We find that the number of residue-residue contacts observed per-residue (contact density) does not distinguish flexible and rigid binding sites, suggesting a role for specific interactions and amino acids in modulating the conformational changes. Examination of hydrogen bonding and hydrophobic interactions reveals that cases that do not undergo conformational change have high polar interactions constituting the binding pockets. Intriguingly, the large, aromatic amino acid tryptophan has a high propensity to occur at the binding sites of examples where a large conformational change has been noted. Further, in large conformational change examples, hydrophobic-hydrophobic, aromatic-aromatic, and hydrophobic-polar residue pair interactions are dominant. Further analysis of the Ramachandran dihedral angles (phi, psi) reveals that the residues adopting disallowed conformations are found in both rigid and flexible cases. More importantly, the binding site residues adopting disallowed conformations clustered narrowly into two specific regions of the L-Ala Ramachandran map. Examination of the dihedral angles changes upon ligand binding shows that the magnitude of phi, psi changes are in general minimal, although some large changes particularly between right-handed alpha-helical and extended conformations are seen. Our work further provides an account of conformational changes in the dihedral angles space. The findings reported here are expected to assist in providing a framework for predicting protein-ligand complexes and for template-based prediction of protein function.

Amino Acids↗

PubMed2006

Computational study of the fibril organization of polyglutamine repeats reveals a common motif identified in beta-helices.

The formation of fibril aggregates by long polyglutamine sequences is assumed to play a major role in neurodegenerative diseases such as Huntington. Here, we model peptides rich in glutamine, through a series of molecular dynamics simulations. Starting from a rigid nanotube-like conformation, we have obtained a new conformational template that shares structural features of a tubular helix and of a beta-helix conformational organization. Our new model can be described as a super-helical arrangement of flat beta-sheet segments linked by planar turns or bends. Interestingly, our comprehensive analysis of the Protein Data Bank reveals that this is a common motif in beta-helices (termed beta-bend), although it has not been identified so far. The motif is based on the alternation of beta-sheet and helical conformation as the protein sequence is followed from the N to the C termini (beta-alpha(R)-beta-polyPro-beta). We further identify this motif in the ssNMR structure of the protofibril of the amyloidogenic peptide Abeta(1-40). The recurrence of the beta-bend suggests a general mode of connecting long parallel beta-sheet segments that would allow the growth of partially ordered fibril structures. The design allows the peptide backbone to change direction with a minimal loss of main chain hydrogen bonds. The identification of a coherent organization beyond that of the beta-sheet segments in different folds rich in parallel beta-sheets suggests a higher degree of ordered structure in protein fibrils, in agreement with their low solubility and dense molecular packing.

Amino Acid Motifs↗

PubMed2006

Sequence and structure analysis of parallel beta helices: implication for constructing amyloid structural models.

Increasing evidence suggests that amyloids and parallel beta helices may share similar motifs. A systemic analysis of beta helices is performed to examine their sequence and structural characteristics. Ile prefers to occur in beta strands. In contrast, Pro is disfavored, compatible with the underlying assumption in Pro-scanning mutagenesis. Cys, Asn, and Phe form significant homostacking (identical amino acid interactions). Asn is highly conserved in the high-energy, left-handed alpha-helical conformation, where it frequently forms amide stacking. Based on the observed prominent stacking of chemically similar residues in parallel beta helices, we propose that within the "cross-beta" framework, amyloids with longer peptide chains may have common structural features of in-register, parallel alignment, with the side chains forming identical amino acid ladders. The requirement of ladder formation limits the combinations of side chain interactions. Such a limit combined with environmental conditions (e.g., pH, concentration) could be a major reason for the ability of most polypeptides to form amyloids.

Amino Acid Sequence↗

PubMed2005

Fibril modelling by sequence and structure conservation analysis combined with protein docking techniques: beta(2)-microglobulin amyloidosis.

Obtaining atomic resolution structural models of amyloid fibrils is currently impossible, yet crucial for our understanding of the amyloid mechanism. Different pathways in the transformation of a native globular domain to an amyloid fibril invariably involve domain destabilization. Hence, locating the unstable segments of a domain is important for understanding its amyloidogenic transformation and possibly control it. Since relative conservation is suggested to relate to local stability, we performed an extensive, sequence and structure conservation analysis of the beta(2)-microglobulin (beta(2)-m) domain. Our dataset include 51 high resolution structures belonging to the "C1 set domain" family and 132 clustered PSI-BLAST search results. Segments of the beta(2)-m domain corresponding to strands A (residues 12-18), D (45-55) and G (91-95) were found to be less conserved and stable, while the central strands B (residues 22-28), C (36-41), E (62-70) and F (78-83) were found conserved and stable. Our findings are supported by accumulating observations from various experimental methods, including urea denaturation, limited proteolysis, H/D exchange and structure determination by both NMR and X-ray crystallography. We used our conservation findings together with experimental literature information to suggest a structural model for the polymerized unit of beta(2)-m. Pairwise protein docking and subsequent monomer stacking in the same manner suggest a fibril model consistent with the cross-beta structure.

Amyloid↗

PubMed2005

Energy landscape of amyloidogenic peptide oligomerization by parallel-tempering molecular dynamics simulation: significant role of Asn ladder.

Recent evidence suggests that amyloidogenic oligomers may be the toxic species in cell cultures. Thus, it is crucial to understand their structure and oligomerization mechanism in atomistic detail. By employing tens of fast central processing units and an advanced phase-space sampling algorithm, parallel-tempering molecular dynamics, we have explored the energy landscape of amyloidogenic peptide oligomerization in explicit water. A pentapeptide, DFNKF, derived from human calcitonin and its mutant, DFAKF, was simulated with a total simulation time of approximately 500 ns. The detailed oligomerization process of a DFNKF parallel beta-sheet formation at 300 K has been characterized. The assembly of a parallel beta-sheet from the amorphous state mainly occurs via a "bottleneck" channel where the interstrand Asn-Asn stacking is the major interaction. The interactions of Asn-Asn stacking include both backbone and side-chain hydrogen bonds. The Asn-Asn interactions work like "glue" by sticking the DFNKF strands together and assist the "on-pathway" oligomerization. The Asn-Asn stacking observed here is similar to the Asn ladder commonly found in globular beta-helix proteins. A control run shows that when Asn is mutated to Ala, the stability and population of the DFAKF parallel beta-sheet is decreased. Furthermore, our in vitro mutagenesis experiments show that the ability of DFAKF peptides to form amyloid fibrils is significantly reduced, in agreement with the simulations. Knowledge of the energy landscape of oligomerization may provide hints for rational drug design, preventing amyloid-associated diseases.

Algorithms↗

PubMed2004

Analysis of ordered and disordered protein complexes reveals structural features discriminating between stable and unstable monomers.

Most proteins exist in the cell as multi-component assemblies. However, which proteins need to be present simultaneously in order to perform a given function is frequently unknown. The first step toward this goal would be to predict proteins that can function only when in a complexed form. Here, we propose a scheme to distinguish whether the protein components are ordered (stable) or disordered when separated from their complexed partners. We analyze structural characteristics of several types of complexes, such as natively unstructured proteins, ribosomal proteins, two-state and three-state complexes, and crystal-packing dimers. Our analysis makes use of the fact that natively unstructured proteins, which undergo a disorder-to-order transition upon binding their partner, and stable monomeric proteins, which exist as dimers only in their crystal form, provide examples of two vastly different scenarios. We find that ordered monomers can be distinguished from disordered monomers on the basis of the per-residue surface and interface areas, which are significantly smaller for ordered proteins. With this scale, two-state dimers (where the monomers unfold upon dimer separation) and ribosomal proteins are shown to resemble disordered proteins. On the other hand, crystal-packing dimers, whose monomers are stable in solution, fall into the ordered protein category. While there should be a continuum in the distributions, nevertheless, the per-residue scale measures the confidence in the determination of whether a protein can exist as a stable monomer. Further analysis, focusing on the chemical and contact preferences at the interface, interior and exposed surface areas, reveals that disordered proteins lack a strong hydrophobic core and are composed of highly polar surface area. We discuss the implication of our results for de novo design of stable monomeric proteins and peptides.

Macromolecular Substances↗

PubMed2004

Modulating functional loop movements: the role of highly conserved residues in the correlated loop motions.

Loop flexibility in enzymes plays a vital role in correctly positioning catalytically important residues. This strong relationship between enzyme flexibility and function provides an opportunity to engineer new substrates and inhibitors. It further allows the design of site-directed mutagenesis experiments to explore enzymatic activity through the control of flexibility of a functional loop. Earlier, we described a novel mechanism in which a small loop triggers the motions of a functional loop in three enzymes (beta-1,4-galactosyltransferase, lipase, and enolase) unrelated in sequence, structure, or function. Here, we further address the question of how the interactions between various flexible loops modulate the movements of the functional loop. We examine beta-1,4-galactosyltransferase as a model system in which a Long loop undergoes a large conformational change (moves in space up to 20 A) upon substrate binding in addition to a small loop (Trp loop) that shows a considerably smaller conformational change. Our molecular-dynamics simulations carried out in implicit and explicit solvent show that, in addition to these two loops, two other neighboring loops are also highly flexible. These loops are in contact with either the Long loop or the Trp loop. Analysis of the covariance of the spatial displacement of the residues reveals that coupled motions occur only in one of these two loops. Sequence analysis indicates that loops correlated in their motions also have highly conserved residues involved in the loop-loop interactions. Further, analysis of crystal structures and simulations in explicit water open the possibility that the Trp loop that triggers the movement of the Long loop in the unbound conformation may also play the same role in the substrate-bound conformation through its contact with the conserved and correlated third loop. Our proposition is supported by the observation that four of the five conserved positions in the third loop are at the interface with the Trp loop. Evolution appears to select residues that drive the functional Long loop to a large conformational change. These observations suggest that altering selected loop-loop interactions might modulate the movements of the functional loop.

Amino Acid Sequence↗

PubMed2004

Sequence and structural analysis of cellular retinoic acid-binding proteins reveals a network of conserved hydrophobic interactions.

Proteins in the intracellular lipid-binding protein (iLBP) family show remarkably high structural conservation despite their low-sequence identity. A multiple-sequence alignment using 52 sequences of iLBP family members revealed 15 fully conserved positions, with a disproportionately high number of these (n=7) located in the relatively small helical region. The conserved positions displayed high structural conservation based on comparisons of known iLBP crystal structures. It is striking that the beta-sheet domain had few conserved positions, despite its high structural conservation. This observation prompted us to analyze pair-wise interactions within the beta-sheet region to ask whether structural information was encoded in interacting amino acid pairs. We conducted this analysis on the iLBP family member, cellular retinoic acid-binding protein I (CRABP I), whose folding mechanism is under study in our laboratory. Indeed, an analysis based on a simple classification of hydrophobic and polar amino acids revealed a network of conserved interactions in CRABP I that cluster spatially, suggesting a possible nucleation site for folding. Significantly, a small number of residues participated in multiple conserved interactions, suggesting a key role for these sites in the structure and folding of CRABP I. The results presented here correlate well with available experimental evidence on folding of CRABPs and their family members and suggest future experiments. The analysis also shows the usefulness of considering pair-wise conservation based on a simple classification of amino acids, in analyzing sequences and structures to find common core regions among homologues.

Amino Acid Sequence↗

PubMed2004

Side chain interactions determine the amyloid organization: a single layer beta-sheet molecular structure of the calcitonin peptide segment 15-19.

In this paper we present a detailed atomic model for a protofilament, the most basic organization level, of the amyloid fibre formed by the peptide DFNKF. This pentapeptide is a segment derived from the human calcitonin, a natural amyloidogenic protein. Our model, which represents the outcome of extensive explicit solvent molecular dynamics (MD) simulations of different strand/sheet organizations, is a single beta-sheet filament largely without a hydrophobic core. Nevertheless, this structure is capable of reproducing the main features of the characteristic amyloid fibril organization and provides clues to the molecular basis of its experimental aggregation behaviour. Our results show that the side chains' chemical diversity induces the formation of a complex network of interactions that finally determine the microscopic arrangement of the strands at the protofilament level. This network of interactions, consisting of both side chain-side chain and backbone-side chain interactions, confers on the final single beta-sheet arrangement an unexpected stability, both by enhancing the association of related chemical groups and, at the same time, by shielding the hydrophobic segments from the polar solvent. The chemical physical characterization of this protofilament provides hints to the possible thermodynamical basis of the supra molecular organization that allows the formation of the filaments by lateral association of the preformed protofibrils. Its regular, highly polarized structure shows how other protofilaments can assemble. In terms of structural biology, our results clearly indicate that an amyloid organization implies a degree of complexity far beyond a simple nonspecific association of peptide strands via amide hydrogen bonds.

Amino Acid Sequence↗

PubMed2004

The stability and dynamics of the human calcitonin amyloid peptide DFNKF.

The stability and dynamics of the human calcitonin-derived peptide DFNKF (hCT(15-19)) are studied using molecular dynamics (MD) simulations. Experimentally, this peptide is highly amyloidogenic and forms fibrils similar to the full length calcitonin. Previous comparative MD studies have found that the parallel beta-stranded sheet is a stable organization of the DFNKF protofibril. Here, we probe the stability and dynamics of the small parallel DFNKF oligomers. The results show that even small DFNKF oligomers, such as trimers and tetramers, are stable for a sufficient time in the MD simulations, indicating that the crucial nucleus seed size for amyloid formation can be quite small. The simulations also show that the stability of DFNKF oligomers increases with their sizes. The small but stable seed may reflect the experimental rapid formation of the DFNKF fibrils. Further, a noncooperative process of parallel beta-sheet formation from the out-of-register trimer is observed in the simulations. In general, the residues of DFNKF peptides near the N-/C-termini are more flexible, whereas the interior residues are more stable. Simulations of mutants and capped peptides show that both interstrand hydrophobic and electrostatic interactions play important roles in stabilizing the DFNKF parallel oligomers. This study provides insights into amyloid formation.

Amyloid↗

PubMed2003

Extended disordered proteins: targeting function with less scaffold.

It has been estimated that a large fraction of cellular proteins are natively disordered. Current opinion largely holds that natively disordered proteins are more 'adaptive', leading to advantages in regulation and in binding diverse ligands. Here, we argue for another, simple, physically based reason. Disordered proteins often have large intermolecular interfaces, the size of which is dictated by protein function. For proteins to be stable as monomers with extensive interfaces, protein size would need to be 2-3 times larger. This would either increase cellular crowding or enlarge the size of the cell by 15-30%, owing to the increase in the sequence length. Smaller sizes of cells, proteins, DNA and RNA conserve energy. Thus, disordered proteins provide a simple yet elegant solution to having large intermolecular interfaces, but with smaller protein, genome and cell sizes.

Models, Molecular↗