title: Influence of a heptad repeat stutter on the pH-dependent conformational behavior of the central coiled-coil from influenza hemagglutinin HA2.
abstract: The coiled-coil is one of the most common protein structural motifs. Amino acid sequences of regions that participate in coiled-coils contain a heptad repeat in which every third then forth residue is occupied by a hydrophobic residue. Here we examine the consequences of a "stutter," a deviation of the idealized heptad repeat that is found in the central coiled-coil of influenza hemagluttinin HA2. Characterization of a peptide containing the native stutter-containing HA2 sequence, as well as several variants in which the stutter was engineered out to restore an idealized heptad repeat pattern, revealed that the stutter is important for allowing coiled-coil formation in the WT HA2 at both neutral and low pH (7.1 and 4.5). By contrast, all variants that contained idealized heptad repeats exhibited marked pH-dependent coiled-coil formation with structures forming much more stably at low pH. A crystal structure of one variant containing an idealized heptad repeat, and comparison to the WT HA2 structure, suggest that the stutter distorts the optimal interhelical core packing arrangement, resulting in unwinding of the coiled-coil superhelix. Interactions between acidic side chains, in particular E69 and E74 (present in all peptides studied), are suggested to play a role in mediating these pH-dependent conformational effects. This conclusion is partially supported by studies on HA2 variant peptides in which these positions were altered to aspartic acid. These results provide new insight into the structural role of the heptad repeat stutter in HA2.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NewYork, 10461.
authors.forename: Chelsea D
authors.initials: CD
authors.lastname: Higgins
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Malashkevich
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Lai
mesh_terms: D020816:Amino Acid Motifs; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006389:Hemagglutinins, Viral / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009980:Influenza A virus / Q000737:chemistry*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics*; D053586:Virus Internalization
chemical_list: D006389:Hemagglutinins, Viral; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus
keywords: influenza; pH-dependent structure; protein design; protein structure; viral membrane fusion
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