PI: Eklund (SLU)
Project: Structure/function studies of virulence associated adhesion organelles from pathogenic Gram negative bacteria
Structure/function studies of virulence associated adhesion organelles from pathogenic Gram negative bacteria
The chaperone/usher pathway constitutes a simple model system to investigate fundamental principles of protein folding, transport, and assembly into cellular organelles. This pathway is used to assemble adhesive organelles in Gram negative pathogens. Adhesive organelles are multimeric protein complexes, often in the form of rod-like fibres called fimbriae or pili that are used for presentation of the receptor-binding adhesin on the bacterial cell surface. These organelles play a crucial role in the pathogenesis of many virulent bacterial strains, as they mediate binding between the invading organisms and complementary receptors on the surfaces of host cells. The chaperone/usher pathway is a highly conserved assembly system consisting of periplasmic chaperones and outer membrane proteins called ushers that operate in pairs. The usher molecules are large pore-forming proteins that are involved in export of organelle subunits and that also function as platforms for organelle assembly. Periplasmic chaperones bind to developing interactive surfaces on nascent subunit proteins to promote release from the inner membrane and proper folding, and to prevent non-productive aggregation in the periplasm. Chaperone/subunit preassembly complexes are targeted to the usher where pilus subunits are dissociated from the chaperone and incorporated into organelles in a defined order.
Structure-function studies of periplasmic chaperones
The structure of the SfaE chaperone from the S pilus system which was solved late 1997 has been refined against five different data sets collected from crystals grown under different conditions. All of these models are essentially identical except that different ionic species appear to be bound to Arg 8 in the subunit binding cleft. This residue is of critical importance for chaperone function since it is involved in anchoring the C-terminus of pilus subunits in the subunit-binding cleft of the chaperone. The structure of the type 1 chaperone FimC has been solved by molecular replacement using a refined SfaE model as the search probe. Comparison of these two chaperones and the PapD chaperone revealed a conserved cluster of water molecules linking Arg 8 in the cleft to the interdomain salt bridge system present in all chaperones of this type. Point mutations of the salt-bridge residues in PapD showed that these are critical for chaperone stability. In addition, the salt bridge system might be involved in release of subunits from the chaperone at the usher site, since one of the residues could be replaced without significant loss of stability or subunit-binding while rendering the chaperone non-functional in pilus assembly.
Structure determination of a chaperone/subunit complex FimC/FimH
The year of 1998 ended on a very happy note with the long awaited structure solution of the preassembly complex between the type 1 pilus chaperone FimC and the mannose-binding adhesin FimH using SeMet MAD data collected at BM14 in Grenoble in December. The solvent flattened MAD phased map showed very clear density for one copy of the complex, for which we have now completed the first model. As expected, a second copy of the complex is present in the asymmetric unit, but density for this complex is of much poorer quality. We have roughly placed a second copy of the CH complex into this density and are about to start refinement of the structure.
FimH, which is the mannose binding adhesin located at the tip of type 1 pili, is folded into two domains; an N-terminal carbohydrate binding domain, and a C terminal pilin domain which is used to anchor the adhesin to the pilus, and which has (limited) sequence homology to the structural pilins of the type 1 pilus. The pilin domain has the same topology as an IgV domain. It can be aligned to the IgC-like N-terminal domain of the FimC chaperone with an r.m.s.d. of 1.9 Å for 64 aligned C[[alpha]] atoms. The chaperone interacts with the pilin almost exclusively via its N-terminal domain, which is the most conserved of the two domains in the chaperone. In the FimH pilin domain, the barrel is open at one end with a gap between the A and F strands. The chaperone, on the other hand, has an additional strand G1 between its A1 and F1 strands. In the complex, the G1 strand of the chaperone inserts between the A and F strands of the pilin domain to make a closed barrel.
The lectin domain of FimH is folded as an elongated 11-stranded, essentially jelly-roll, sandwich. The connection between the pilin and lectin domains is though an extended piece of chain with essentially no contacts between the two domains. Thus, in solution, or in the pilus, the lectin domain would probably be free to move around substantially.
The structure determination of the FimC-FimH complex represents a major breakthrough in our understanding of pilus biogenesis and pilus-mediated adhesion. It is the first structure of a chaperone-subunit complex to be solved, providing detailed information about the interaction between a periplasmic immunoglobulin-like chaperone and a target pilin subunit. No structure of a pilin or adhesin subunit has previously been determined; the FimC-FimH structure is thus also the first structure of a structural pilus subunit (represented by the FimH pilin domain) and of a bacterial adhesin (the FimH lectin domain). In addition to giving insight into the principles of pilus structure and assembly, the FimC-FimH structure will facilitate the development of novel antibacterial vaccines and chaperone inhibitors. In the face of rising antibiotic resistance it is not too farfetched to say that this would be of some importance.
Publications
Soto GE, Dodson KW, Ogg D, Liu C, Heuser J, Knight S, Kihlberg J, Jones CH & Hultgren SJ (1998): Periplasmic Chaperone Recognition Motif of Subunits Mediates Quaternary Interactions in the Pilus. EMBO J. 17:6155-6167.
Hung DL, Knight SD & Hultgren SJ (1999): Probing conserved surfaces on PapD. Mol Micro, in the press.
Hung DL, Pinkner JS, Hultgren SJ & Knight SD (1999): Structural Basis of Chaperone Self-Capping in P Pilus Biogenesis. Submitted to PNAS.
Hultgren SJ, Hung DL, Jones CH & Knight S (1998). Periplasmic PapD-Like Chaperones in Bacteria: Structure and Function. In Molecular Biology of Chaperones (edt Bernd Bukau), Harwood Academic Publishers, Chur.
Latest update at 18 February, 1999.