Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity

Abstract

Influenza virus neuraminidase (iNA) is a homotetrameric surface protein of the influenza virus and an established target for antiviral drugs. In contrast to neuraminidases (NAs) of other biological systems (non-iNAs), enzymatic activity of iNA is only observed in a quaternary assembly and iNA needs the tetramerization to mediate enzymatic activity. Obviously, differences on a molecular level between iNA and non-iNAs are responsible for this intriguing observation. Comparison between protein structures and multiple sequence alignment allow the identification of differences in amino acid composition in crucial regions of the enzyme, such as next to the conserved D151 and the 150-loop. These differences in amino acid sequence and protein tetramerization are likely to alter the dynamics of the system. Therefore, we performed molecular dynamics simulations to investigate differences in the molecular flexibility of monomers, dimers, and tetramers of iNAs of subtype N1 (avian 2004, pandemic 1918 and pandemic 2009 iNA) and as comparison the non-iNA monomer from Clostridium perfringens. We show that conformational transitions of iNA are crucially influenced by its assembly state. The protein–protein interface induces a complex hydrogen-bonding network between the 110-helix and the 150-loop, which consequently stabilizes the structural arrangement of the binding site. Therefore, we claim that these altered dynamics are responsible for the dependence of iNA’s catalytic activity on the tetrameric assembly. Only the tetramerization-induced balance between stabilization and altered local flexibility in the binding site provides the appropriate arrangement of key residues for iNA’s catalytic activity.

Biological and Protective Properties of Immune Sera Directed to the Influenza Virus Neuraminidase

“…The expression of NA in the native conformation is believed to be crucial for the induction of anti-NA antibodies with inhibitory activity (62). As only the native tetrameric NA is enzymatically active (63,64), sialidase activity is regarded as an indicator of NA immunogenicity (62). In contrast to vaccination with VRPs, intramuscular immunization of chickens with inactivated influenza virus resulted in significantly lower titers of anti-NA antibodies, although relatively high anti-HA titers were induced.…”

Dynamic regulation of phenylalanine hydroxylase

“…Enzyme regulation involving changes in assembly state has already been established for several enzymatic systems including influenza neuraminidase. Molecular dynamics simulations were found to be helpful in the characterization of the changes in the system's dynamics after the formation of additional macromolecular interfaces by tetramerization [70].…”

Impact of tetramerization on the ligand recognition of N1 influenza neuraminidase via MMGBSA approach

“…From the pair of interactions present in most of the apo and holo tetrameric closed‐ and open‐loop, it is observed that five pairs of polar interactions: Arg107.NH1(chain A)–Gln.O136(chain C), Ser110.OG(chain A)–Asp142.OD1(chain C), Tyr169.OH(chain A)–Asp113.N(chain D), Asp113.N(chain B)–Tyr169.OH(chain D) and Tyr169.OH(chain B)–Asp113.N(chain C), and three pair of hydrophobic interactions: Ile108.CD1(chain A)–Phe115.CE2(chain C), Ile108.CG1(chain A)–Leu139.CD1(chain C) and Trp458.CD1(chain A)–Val176.CG1(chain C) are present in most complexes. From these pairs of interactions, Arg107.NH1(chain A)–Gln.O136(chain C) and Ser110.OG(chain A)–Asp142.OD1(C) have also been observed for other tetramers of subtype N1 (avian 2004, pandemic 1918, and pandemic 2009 influenza NA) through MD simulations . These interactions are localized between the α‐helix around S105‐S110 and the 150‐loop (135‐155), from which two residues (Arg151 and Arg152) are present in the 150‐loop and participate in oseltamivir or sialic acid stabilization (Figures ); therefore, differences in the energetic state of this interaction together with the other seven protein‐protein interactions may impact the affinity of ligands in the active site of tetrameric NA.…”

“…Recently, von Grafensteina et al . revealed that the altered dynamics of the 150‐loop depend on the assembly state . The tetrameric open‐loop and closed‐loop NA in the free and bound state was used to show how the conformational differences in the loop and apo and bound state impact the map of interaction on a tetrameric system.…”

“…More recently, von Grafenstein et al . investigated differences in the molecular flexibility of monomers, dimers, and tetramers of NAs of subtype N1 (avian 2004, pandemic 1918, and pandemic 2009 iNA), identifying that the protein–protein interface induces the formation of a pair of polar interactions between the 110‐helix and the 150‐loop, which stabilizes the binding site . Although these observations suggest a stabilizing mechanism between subunits and differences in the binding mechanism between the four subunits, the structural and energetic details of tetrameric stabilization, ligand binding affinity and structural differences between the different subunits has not been elucidated to date.…”