Thursday, 2 June 2011

The Structural Biology of Pilus Biosynthesis: Or, How Bacteria Man the Pumps

Science Week at Birkbeck College was celebrated for the second time in early May 2011 with presentations from each of the college’s three science departments. The two lectures from researchers in Biological Sciences were linked by the common theme of nano-machines in biology. Just as a car engine, for example, is built up from many interacting parts, so some proteins work together in large complexes to do particular jobs within cells. Professor Helen Saibil, whose ground-breaking work in electron microscopy has featured earlier in this blog (see November 2010 post) presented some of her research into chaperones, protein machines that carry out “quality control” work enabling other proteins to form into and stay in the precise three-dimensional shapes they need to function.

The other speaker from Biological Sciences was Professor Gabriel Waksman, a distinguished structural biologist who combines a very successful research career with running both Birkbeck’s Biological Sciences department and the Research Department of Structural and Molecular Biology at UCL. Professor Waksman’s work for many years has focused on the complex structures through which bacteria interact with the outside world. Pathogenic bacteria cause problems for their hosts only when they interact with them, by secreting toxic substances into their environment or attaching to host cells. Now, when bacteria are rapidly developing resistance to many traditional antibiotics and more antibiotics, particularly with novel mechanisms of action, are desperately needed, some of these mechanisms are at last becoming understood.

Gram negative bacteria, which have double cell walls, often carry hair-like fibres or filaments known as pili on their surfaces. Some bacteria use these to bind to receptors on the surfaces of host cells, a process that can trigger the host cell surrounding and engulfing the bacteria in infection. Different forms of bacteria even from the same species carry different pili that bind to different cell receptors: for example, some E. coli bind to and infect bladder cells causing cystitis, while others infect kidney cells causing pyelonephritis. E. coli can also use pili to bind to each other, forming colonies around the bladder that are particularly difficult to treat.

The type of E. coli that infects the kidney carries a large number of so-called P pili on its outer membrane. These consist of a relatively thick rod near the cell wall and a thin filamentous tip. The whole pilus is made up of thousands of similar protein subunits encoded by genes within the Pap gene cluster. Almost all are identical PapA subunits, and these form the rod: the tip consists of just a few homologous PapE subunits, tipped by the sensor, PapG, which recognises and binds to kidney cells.

Pilus subunits, like all proteins, are synthesised in the cytoplasm; they need to be transported to the outer membrane and polymerise to form the pilus structure, and this complex task is achieved by other Pap proteins. When a pilus subunit is synthesised it is first translocated across the inner membrane into the periplasm, where it will be immediately degraded unless it can bind to a PapD protein. This acts as a chaperone, moving the subunit to the outer membrane where it docks with the membrane-bound PapC. This latter, or “usher” protein, is the core of the pilus biosynthesis molecular machine, and PapC and PapD together give the process its name: the chaperone-usher pathway. PapC has a large central pore through which the intact pilus is secreted.

Over many years, Gabriel Waksman and his group have solved the structure of many of these Pap proteins, and they have now built up an accurate picture at atomic resolution of how the chaperone-usher pathway works. The first structure to be solved was that of a binary complex of one pilus subunit, PapK, bound to the chaperone PapD (PDB 1PDK). PapK – and, subsequently, each of the pilus subunits – was found to have an immunoglobulin type fold, but with one beta-strand missing. This structure can only be stabilised when another protein, either the chaperone or a second pilus subunit, completes this fold with a strand of its own. One by one, starting with the tip subunit PapG, chaperone-subunit complexes migrate to the PapC usher, where the chaperone strand is replaced by a strand from another subunit in what has been termed a “donor strand exchange” model of polymerisation. The pilus fibre therefore forms from a series of “typical” immuno-globulin-like subunits in which each subunit is completed with a single strand from the next nearest subunit. The pilus biogenesis process only stops when a PapH, or “terminator” subunit is incorporated.

The usher forms a wide pore in the outer membrane (PDB 2VQI) and acts both to synthesise and to secrete the pilus polymer. It is a long, multi-domain protein. Using X-ray crystallography, Waksman’s group first determined that the pore comprises a very large beta barrel derived from the central domain of the usher, with a small sub-domain embedded within this domain forming a “plug” that blocks the pore when it is not being used. The usher also has a short N-terminal domain that dangles down into the periplasm and grabs on to chaperone-subunit complexes. The function of the C-terminal domain, however, remained unknown until the group solved the structure of an intact usher-chaperone-subunit complex.

It was only when this intact structure was solved that Waksman’s group really began to understand the mechanism of this complex “molecular machine”. For this, the group used a homologous system in which the usher is a protein known as FimD, the chaperone is FimC and the bound subunit FimH. This structure was a “first” in several ways, not least because it was the first time that an intact, folded protein was observed inside the pore of another protein structure. In this structure, the C-terminal domain of the FimD usher was seen to bind to the chaperone-subunit complex. It appears that, once the N-terminal usher domain has grabbed on to a chaperone-subunit complex and moved it into the usher, that complex will move up the usher structure to the binding site on the C-terminal domain, freeing the N-terminal domain to capture the next subunit.

This work, the culmination of fifteen years’ study of this secretion system, has just appeared in Nature (published online ahead of print 1 June 2011). More importantly, however, this elegant piece of structural biology may be exploited in the war against bacterial infection. A drug that bound to the usher and prevented pilus biosynthesis – a “pilicide” – would not kill the bacteria, but it would prevent them from binding to their target cells and also from forming the antibiotic-resistant colonies that can remain in the urinary tract for years and that lead to persistent infection.