Phage display and crystallographic analysis reveals potential substrate/binding site interactions in the protein secretion chaperone CsaA from Agrobacterium tumefaciens.
The protein CsaA has been proposed to function as a protein secretion chaperone in bacteria that lack the Sec-dependent protein-targeting chaperone SecB. CsaA is a homodimer with two putative substrate-binding pockets, one in each monomer. To test the hypothesis that these cavities are indeed substrate-binding sites able to interact with other polypeptide chains, we selected a peptide that bound to CsaA from a random peptide library displayed on phage. Presented here is the structure of CsaA from Agrobacterium tumefaciens (AtCsaA) solved in the presence and absence of the selected peptide. To promote co-crystallization, the sequence for this peptide was genetically fused to the amino-terminus of AtCsaA. The resulting 1.65 A resolution crystal structure reveals that the tethered peptide from one AtCsaA molecule binds to the proposed substrate-binding pocket of a symmetry-related molecule possibly mimicking the interaction between a pre-protein substrate and CsaA. The structure shows that the peptide lies in an extended conformation with alanine, proline and glutamine side chains pointing into the binding pocket. The peptide interacts with the atoms of the AtCsaA-binding pocket via seven direct hydrogen bonds. The side chain of a conserved pocket residue, Arg76, has an "up" conformation when the CsaA-binding site is empty and a "down" conformation when the CsaA-binding site is occupied, suggesting that this residue may function to stabilize the peptide in the binding cavity. The presented aggregation assays, phage-display analysis and structural analysis are consistent with AtCsaA being a general chaperone. The properties of the proposed CsaA-binding pocket/peptide interactions are compared to those from other structurally characterized molecular chaperones.
Feldman
Anat R
AR
Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, Canada.
Shapova
Yuliya A
YA
Wu
Sampson S T
SS
Oliver
David C
DC
Heller
Markus
M
McIntosh
Lawrence P
LP
Scott
Jamie K
JK
Paetzel
Mark
M
D016960:Agrobacterium tumefaciens / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000737:chemistry
D001426:Bacterial Proteins; D008024:Ligands; D018832:Molecular Chaperones; D019151:Peptide Library; D010455:Peptides