Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

Morgan Lawrenz,J Andrew Mccammon,Jeff Wereszczynski,Rommie Amaro,Ross Walker,Adrian Roitberg

doi:10.1002/prot.22761

Abstract

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium-bound and calcium-free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley-Liss, Inc.

Highlights

Type A influenza virus is becoming a worldwide pandemic threat due to its virulence and transmissibility in people.* The viral surface protein neuraminidase, along with hemagglutinin, classifies influenza subtypes and fulfills an important role in viral propagation by cleaving a terminal sialic acid from host cell surfaces; neuraminidase is a key drug target for controlling flu infection
The structure of the N1 calcium binding site includes close ion contacts with the backbone carbonyls of D293, N294, G297, G345, A346, Y347, and the carboxyl groups of both D293 and D324, with similar interactions observed in group 1 and 2 crystal structures.[1,2,3,4,9]
Removal of the ion can be expected to destabilize these residues and the loops on which they are located, leading to increased fluctuation. Both GROMOS96 and AMBER FF99SB ion-bound molecular dynamics (MD) simulations were checked for similar root mean squared fluctuation (RMSF) per residue Ca (Supporting Information Fig. 1), with the most flexibility observed for

Summary

Introduction

Type A influenza virus is becoming a worldwide pandemic threat due to its virulence and transmissibility in people.* The viral surface protein neuraminidase, along with hemagglutinin, classifies influenza subtypes and fulfills an important role in viral propagation by cleaving a terminal sialic acid from host cell surfaces; neuraminidase is a key drug target for controlling flu infection. The enzyme has two distinct families,[1] group 1 (N1, N4, N5 and N8) and group 2 (N2, N3, N6 and N7), with group 1 including the N1 subtype found in recent outbreaks of the highly pathogenic subtype H5N1, as well as the 1918 epidemic and ‘‘swine flu’’ H1N1. The protein exhibits a high rate of antigenic shift and drift, yielding proteins with heterogeneous sequences that can reduce sensitivity to the clinically used inhibitors oseltamivir (marketed as Tamiflu by Roche Pharmaceuticals, Basel, Switzerland), zanamivir (marketed as Relenza by GlaxoSmithKline, Bentford), and peramivir (BioCryst Pharmaceuticals, Birmingham, AL). The specific role of this calcium ion is unknown, but experiments have supported its importance for wild-type enzyme activity[6] and enzyme

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Conclusion