Protein-dynamics of the putative HCV receptor CD81 large extracellular loop

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Abstract

The human CD81 protein is a likely receptor for the binding of hepatitis C virus (HCV) to hepatocytes and therefore a possible target for novel anti-HCV drugs. The two published X-ray structures of the HCV binding region of CD81 (1G8Q and 1IV5) have, particularly in a substructure that is formed by two helices, a slightly different conformation. The abovementioned substructure is a candidate target region for virtual screening approaches. We present here a molecular dynamics study of the two X-ray structures. Our results indicate that the conformation of the two helical regions in one of the X-ray structures (1G8Q) is affected by crystallographic contacts and most likely does not represent the native state of the protein.

Molecular dynamics simulations of the putative HCV receptor CD81 indicate that the conformation of the two helical regions in one of the published X-ray structures is affected by crystallographic contacts and most likely does not represent the native state of the protein. The implications of this result for drug design projects aimed at the CD81/E2 interaction are discussed.

Introduction

Hepatitis C virus (HCV), a positive-stranded RNA virus, is the infectious agent responsible for one subtype of viral hepatitis. Today, 300 million people worldwide are infected by HCV. The therapy of choice currently consists of a combination of ribavirin and (pegylated) interferon, but this therapeutic regimen is expensive and fails in about 40% of all cases.1 Novel therapeutical strategies are therefore urgently needed.

The HCV genome encodes for a single polyprotein of approximately 3000 amino acids. The polyprotein is processed by host and viral peptidases into different viral proteins.2 Among these proteins, the envelope glycoprotein E2 is supposed to be the key protein for viral entry.

Putative receptors for HCV–E2 binding interaction are the human CD81 receptor,3, 4, 5 the low-density lipoprotein receptor (LDLR),6 the human scavenger receptor class B type I7 and liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN).8

Among these receptors, X-ray structures are available for the large extracellular loop (LEL) of CD81 only. Furthermore, the interaction sites with the E2 protein have been identified recently.3, 9 This makes the LEL on CD81 a potential drug target. Recent results by VanCompernolle10 and by our own group (unpublished data) indicate that small molecules which interfere with the CD81/E2 interaction can inhibit the binding of HCV to their host cells.

The three-dimensional structure of LEL was determined by Kitadokoro et al. using X-ray crystallography and two different crystallographic forms (monoclinic: pdb ID 1G8Q and hexagonal: pdb ID 1IV5).11, 12 An important difference between the two structures is a cleft-like motif formed by the C and D helices in the 1G8Q structure which does not appear in the 1IV5 structure. Therefore, different classes of inhibitors for the E2–CD81 interaction process seem to be possible: In the case of the 1G8Q structure, small organic molecules fit into the abovementioned cleft, whereas in the 1IV5 form only surface interactions between the protein and the ligand are possible. Here, we investigate the dynamics of the LEL region of the CD81 receptor by molecular dynamics simulations to gain deeper insight in the CD81–LEL drug target. Our main objective was to determine the physiologically relevant conformation of the LEL region, which can be used as the target protein in virtual high-throughput screening.

Section snippets

Methods

X-ray structures of the monoclinic (pdb ID: 1G8Q) and hexagonal (pdb ID: 1IV5) crystallization form of the CD81-LEL were obtained from the Brookhaven Protein Databank (PDB). All molecular dynamic simulations were performed by the AMBER 7 suite of programs.13 Water molecules were removed from the PDB files and missing protein atoms were added. Disulfide bond formation and charge neutralization (addition of Na+− ions) were carried out using the AMBER utility Xleap. Afterwards, the simulation

Molecular dynamics simulations

CD81–LEL was crystallized as dimer. Each monomer consists of five helices (A–E) where helices A and B provide the main dimer association interface. The E2 binding region is located close to the C and D helices. Therefore, the structures of the C and D helices are important for E2 and inhibitor binding. To evaluate their dynamic behavior, we calculated the average mass-weighted fluctuations of all amino acids of both LEL structures. The calculation was performed on the last 400 ps of the MD

Conclusions

The simulations have, combined with the analysis of the X-ray structures, shown that crystallographic contacts cause a distortion of the CD81–LEL folding in one of the two X-ray structures. Under physiological conditions, CD81–LEL most likely exists in the conformation with a closed cleft between the C and D helices. However, the two helices experience a certain degree of flexibility, and it may be possible to identify—for example, by virtual screening—small molecules that fit into the cleft

Supplementary Material

Supplementary material

Acknowledgements

We appreciate financial support by the German ‘Fonds der Chemischen Industrie’ (FCI).

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Supplementary data associated with this article can be found, in the online version at, doi: 10.1016/j.bmcl.2004.01.036

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