Characterization of de novo four-helix bundles by molecular dynamics simulations

We have investigated the structure and dynamics of three cavitand-based four-helix bundles (caviteins) by computer simulation. In these systems, designed de novo, each of the four helices contain the identical basis sequence EELLKKLEELLKKG (N1). Each cavitein consists of a rigid macrocycle (cavitand) with four aryl linkages, to each of which is connected an N1 peptide by means of a linker peptide. The three caviteins studied here differ only in the linker peptide, which consist of one, two, or three glycine residues. Previous experimental work has shown that these systems exhibit very different behavior in terms of stability and oligomerization states despite the small differences in the linker peptide. Given that to date no three-dimensional structure is available for these caviteins, we have undertaken a series of molecular dynamics (MD) simulations in explicit water to try to rationalize the large differences in the experimentally observed behavior of these systems. Our results provide insight, for the first time, into why and how the cavitein with a single glycine linker forms dimers. In addition, our results indicate why although the two- and three-glycine-linked caviteins have similar stabilities, they have different native-like characteristics: the cavitein with three glycines can form a supercoiled helix, whereas the one with two glycines cannot. These findings may provide a useful guide in the rational de novo design of novel proteins with finely tunable structures and functions in the future.     

Structure and dynamics of de novo proteins from a designed superfamily of 4-helix bundles

Libraries of de novo proteins provide an opportunity to explore the structural and functional potential of biological molecules that have not been biased by billions of years of evolutionary selection. Given the enormity of sequence space, a rational approach to library design is likely to yield a higher fraction of folded and functional proteins than a stochastic sampling of random sequences. We previously investigated the potential of library design by binary patterning of hydrophobic and hydrophilic amino acids. The structure of the most stable protein from a binary patterned library of de novo 4-helix bundles was solved previously and shown to be consistent with the design. One structure, however, cannot fully assess the potential of the design strategy, nor can it account for differences in the stabilities of individual proteins. To more fully probe the quality of the library, we now report the NMR structure of a second protein, S-836. Protein S-836 proved to be a 4-helix bundle, consistent with design. The similarity between the two solved structures reinforces previous evidence that binary patterning can encode stable, 4-helix bundles. Despite their global similarities, the two proteins have cores that are packed at different degrees of tightness. The relationship between packing and dynamics was probed using the Modelfree approach, which showed that regions containing a high frequency of chemical exchange coincide with less well-packed side chains. These studies show (1) that binary patterning can drive folding into a particular topology without the explicit design of residue-by-residue packing, and (2) that within a superfamily of binary patterned proteins, the structures and dynamics of individual proteins are modulated by the identity and packing of residues in the hydrophobic core.     

Structural refinement of the hERG1 pore and voltage‐sensing domains with ROSETTA‐membrane and molecular dynamics simulations

The hERG1 gene (Kv11.1) encodes a voltage‐gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template‐driven de‐novo design with ROSETTA‐membrane modeling using side‐chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2‐Kv2.1 chimera channels, the missing parts are modeled de‐novo. The impact of several alignments on the structure of the S4 helix in the voltage‐sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage‐sensor domain; and the topology of the extracellular S5‐pore linker compared with that established by toxin foot‐printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma