pH‐Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain

• Published in: Journal of molecular biology Vol. 422; no. 4; pp. 477 - 487
• Main Authors: Jaudzems, Kristaps, Askarieh, Glareh, Landreh, Michael, Nordling, Kerstin, Hedhammar, My, Jörnvall, Hans, Rising, Anna, Knight, Stefan D., Johansson, Jan
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 28.09.2012
• Subjects: Amino Acid Sequence; Animals; Araneae; crystal structure; Crystallography, X-Ray - methods; dimerization; Fibroins - chemistry; Fibroins - metabolism; fluorescence; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy - methods; Medicin och hälsovetenskap; Molecular Sequence Data; NMR structure; nuclear magnetic resonance spectroscopy; protein assembly; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; proteins; silk; Silk - chemistry; Silk - metabolism; silk glands; sodium chloride; Spiders - metabolism; spidroin; Structural Biology; Strukturbiologi; tryptophan; Tryptophan - chemistry; Tryptophan - metabolism; X-ray structure; 3D; NMR structure; NOE; MS; NOESY; HSQC; HDX; wt; X-ray structure; protein assembly; spidroin; PDB
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2012.06.004

Formation of spider silk from its constituent proteins—spidroins—involves changes from soluble helical/coil conformations to insoluble β-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild‐type (wt) E. australis NT at pH7.2 and 300mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5–6Å into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH7 and pH6 and indicate that the biological function of NT depends on conversion between the two conformations. [Display omitted] ► Structures of the N-terminal domain (NT) of spider silk proteins are determined. ► NT monomers have a wedged tryptophan side chain that prevents dimerization. ► Our results have implications for the regulation of spider silk formation.