Structure of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus in Complex with a Protective Monoclonal Antibody

Abstract

The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. IMPORTANCEM2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2. Matrix protein 2 (M2) is a structural protein of influenza A viruses and plays an important role in the virus life cycle. This type III membrane protein of 96 amino acid residues has an N-terminal ectodomain (M2e) of 23 residues, a transmembrane domain of 19 residues, and a cytoplasmic domain of 54 residues (1). M2 is classified as a viroporin. Mutational analysis and crystal and nuclear magnetic resonance (NMR) structural analysis of the M2 transmembrane domain revealed that it is composed of a fourstranded coiled coil and that two conserved amino acid residues (M2-His37 and -Trp41) have a key role in acid-induced proton gating (2-5). Following endocytosis of influenza A virions, the acidic endosomal environment activates the M2 channel so that protons enter the virion interior. The resulting acidification loosens the viral ribonucleoprotein complexes from matrix protein 1 (M1), which facilitates their migration through the membrane fusion pore into the host cell cytoplasm (6, 7). M2 can activate the inflammasome, impairs autophagosome maturation, and was recently shown to recruit parts of the autophagosome machinery to sites of virus budding by a conserved motif in its cytoplasmic domain (8, 9).The sequence of M2e is conserved and therefore has frequently been explored for the development of a universal influenza A vaccine (10-13). Immune protection by M2e-directed vaccines has been documented extensively in experimental animal models, including mice, ferrets, and swine (11-13). In addition, a phase I clinical study showed that M2e-based vaccines are safe and immunogenic in humans (10,14). Seasonal influenza A virus infection induces a poor immune responses to M2e, but this weak re...

Crystal Structure of the Conserved Amino Terminus of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus Gripped by an Antibody

“…The first nine amino acid residues of M2 are extremely conserved across all reported influenza A viruses. Human and murine M2e-specific MAbs that recognize different parts of M2e have been described, but how these interact with M2e is largely unknown (4)(5)(6). Here, we report the crystal structure of M2e in complex with a Fab fragment from a MAb that binds to the highly conserved N-terminal part of M2.…”

“…3) (6). The N-terminal ␤-turn from Ser2 to Glu6 that determines M2e binding to MAb148 is absent in the MAb65 complex (Fig.…”

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

“…In addition, AA residues participating in the binding energy were shown to be mainly aromatic residues, as well as Gly or Ser . In several works, remarkable conformational changes in the structure of Abs and antigen during the formation of their complexes were observed …”

Viral channel forming proteins — How to assemble and depolarize lipid membranes in silico

“…Nevertheless most of the structural information is limited to the TMDs of the VCPs and in the case of Vpu it includes also solely the cytoplasmic domain. Most recently a synthetic peptide corresponding to the extramembrane N terminal side of M2 has been crystallized in contact with a monoclonal antibody [17]. The structure can be regarded as a good approximation to the 'in vivo' structure of M2.…”