Dataset Information

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

ABSTRACT: Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1?/CC-Chemokine Ligand 4 (MIP-1?/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 ?s molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1?. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1?, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1?. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1? interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

SUBMITTER: Nguyen AF

PROVIDER: S-EPMC5578167 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Nguyen Anna F AF Kuo Nai-Wei NW Showalter Laura J LJ Ramos Ricardo R Dupureur Cynthia M CM Colvin Michael E ME LiWang Patricia J PJ

International journal of molecular sciences 20170816 8

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from <i>Escherichia coli</i> (<i>E. coli</i>.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II ...[more]

PMID: 28813018

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Project description:Studies aimed at elucidating the reaction mechanism of farnesyltransferase (FTase), which catalyzes the prenylation of many cellular signaling proteins including Ras, has been an active area of research. Much is known regarding substrate binding and the impact of various catalytic site residues on catalysis. However, the molecular level details regarding the conformational rearrangement of farnesyldiphosphate (FPP), which has been proposed via structural analysis and mutagenesis studies to occur prior to the chemical step, is still poorly understood. Following on our previous computational characterization of the resting state of the FTase ternary complex, the thermodynamics of the conformational rearrangement step in the absence of magnesium was investigated for the wild type FTase and the Y300Fbeta mutant complexed with the peptide CVIM. In addition, we also explored the target dependence of the conformational activation step by perturbing isoleucine into a leucine (CVLM). The calculated free energy profiles of the proposed conformational transition confirm the presence of a stable intermediate state, which was identified only when the diphosphate is monoprotonated (FPP2-). The farnesyl group in the computed intermediate state assumes a conformation similar to that of the product complex, particularly for the first two isoprene units. We found that Y300beta can readily form hydrogen bonds with either of the phosphates of FPP. Removing the hydroxyl group on Y300beta does not significantly alter the thermodynamics of the conformational transition, but shifts the location of the intermediate farther away from the nucleophile by 0.5 A, which suggests that Y300beta facilitate the reaction by stabilizing the chemical step. Our results also showed an increased transition barrier height for CVLM (1.5 kcal/mol higher than that of CVIM). Although qualitatively consistent with the findings from the recent kinetic isotope experiments by Fierke and co-workers, the magnitude is not large enough to affect the rate-limiting step.

Dataset Information

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Publications

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

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