Structural studies of the Dickeya dadantii type II secretion system protein GspB.

Abstract

The type II secretion system (T2SS) is the major terminal branch of the general secretory pathway in Gram negative bacteria. It is composed of an outer-membrane secretin, periplasmic pilin-like subunits extending from the inner and outer-membranes, an inner-membrane platform, and an associated cytoplasmic ATPase. The secretin is the only essential component in the outer membrane and is formed by a dodecameric torus of protein subunits. Secreted enzymes and virulence factors pass through the gated pore in the centre of the toroidal secretin dodecamer and out into the extra cellular milieu. In some species of bacteria there are auxiliary proteins that are important for correct assembly and formation of a functional T2SS. One such protein is GspB which, in the plant pathogen D. dadantii, aids transport of the secretin from the inner-membrane, through the periplasm and on to the outer-membrane. In the absence of GspB, the secretin miss-locates to the inner membrane and a functional T2SS is not assembled. GspB is one of four inner-membrane proteins of the T2SS that have a single membrane-spanning helix and periplasmic domain, the others are GspC, GspL and GspM. The hypothesis is therefore that the periplasmic domain of GspB reaches out from the inner-membrane platform into the peptidoglycan-filled periplasm to facilitate the transport of the secretin subunits to the outer-membrane or to stabilize the secretin subunits once they have reached the outer-membrane. In this Thesis, I report the crystal structure of the periplasmic domain of the auxiliary protein GspB. The structure is similar to the homology region of one of the other four single transmembrane helix proteins from the inner-membrane platform, GspC. The homology region domain of GspC had been previously shown to interact with the N-terminal domain of the secretin subunit and these observations suggests that GspB may have taken over or may augment the function of GspC. Here, NMR spectroscopy was used to confirm an interaction between GspB and the N-terminal secretin domain and a model of the interaction is proposed on the basis of the available spectroscopic and biochemical measurements.