A hinge region cis-proline in ribonuclease A acts as a conformational gatekeeper for C-terminal domain swapping.
Domain swapping, the process in which a structural unit is exchanged between monomers to create a dimer containing two subunits of the monomeric fold, is believed to be an important mechanism for oligomerization and the formation of amyloid fibrils. Structural studies have implicated proline as an important residue for domain swapping due to its increased frequency in hinge regions preceding swapped arms. We hypothesized that proline's unique ability to populate both cis and trans peptide bond conformations may allow proline to act as a conformational gatekeeper, regulating interconversion between monomer and domain-swapped dimer forms. The hinge region of RNase A contains a proline at residue 114 that adopts a cis conformation in the monomer and extends to a trans conformation in the C-terminal domain-swapped dimer. Substitution of P114 with residues that strongly prefer a trans peptide bond (Ala, Gly) results in significant population of the C-terminal domain-swapped dimer under near-physiological conditions (pH 8.0, 37 degrees C). This is in stark contrast to dimerization of wild-type RNase A, which requires incubation under extreme conditions such as lyophilization from acetic acid or elevated temperature. In addition, we observe similar results when cis-P114 is mutated to glycine in a homologous RNase, human pancreatic RNase 1. Our results suggest that isomerization at P114 may facilitate population of a partially unfolded intermediate or alternative structure competent for domain swapping and provide support for a more general role for proline isomerization as a conformational gatekeeper in domain swapping and oligomerization.
Miller
Katherine H
KH
Institute for Quantitative Biosciences-Berkeley, University of California, Berkeley, CA 94720-3220, USA.
Karr
Jessica R
JR
Marqusee
Susan
S
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D019281:Dimerization; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011392:Proline; D012259:Ribonuclease, Pancreatic
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