Structural analysis of substrate binding by the TatBC component of the twin-arginine protein transport system

Abstract

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. In Escherichia coli substrate proteins initially bind to the integral membrane TatBC complex which then recruits the protein TatA to effect translocation. Overproduction of TatBC and the substrate protein SufI in the absence of TatA led to the accumulation of TatBC-SufI complexes that could be purified using an affinity tag on the substrate. Three-dimensional structures of the TatBC-SufI complexes and unliganded TatBC were obtained by single-particle electron microscopy and random conical tilt reconstruction. Comparison of the structures shows that substrate molecules bind on the periphery of the TatBC complex and that substrate binding causes a significant reduction in diameter of the TatBC part of the complex. Although the TatBC complex contains multiple copies of the signal peptide-binding TatC protomer, purified TatBC-SufI complexes contain only 1 or 2 SufI molecules. Where 2 substrates are present in the TatBC-SufI complex, they are bound at adjacent sites. These observations imply that only certain TatC protomers within the complex interact with substrate or that there is a negative cooperativity of substrate binding. Similar TatBC-substrate complexes can be generated by an alternative in vitro reconstitution method and using a different substrate protein.membrane protein ͉ Escherichia coli ͉ blue native PAGE ͉ single particle electron microscopy

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

“…This finding is consistent with previous observations that these two proteins copurify in detergent solution as a large TatBC complex and that the size and composition of the complex are not significantly altered by substrate binding (19,22,45,49,50). It is also consistent with the observation that TatBC complexes do not exhibit the exchange of TatC subunits that would be expected if TatC undergoes assembly cycles (23).…”

“…Tat-signal peptides are recognized at the membrane by a TatBC receptor complex (18)(19)(20)(21)(22)(23). Substrate protein binding to TatBC causes a PMF-dependent recruitment of TatA (and presumably TatE) to assemble the active translocation site (18,24).…”

Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

“…There is clear evidence that, in both plant thylakoid and E. coli systems, the TatABC complex has a role in substrate binding. These subunits have been shown to cross-link to in vitro-synthesized substrates under conditions that promote binding but not translocation (9), and a TatBC complex has been purified with one or two substrate molecules bound to it after overexpression of substrates in the absence of TatA (38). In contrast, the TatA complex appears only to become involved after this initial substrate binding step (12).…”

Early Contacts between Substrate Proteins and TatA Translocase Component in Twin-arginine Translocation

“…TatB (1)(2)(3)(4) and TatC (1,5,6) each have the propensity to individually homo-oligomerize. Together they form multimers of functional hetero-dimeric core units (1,4,(7)(8)(9), which when purified from bacteria often also contain some TatA (1,10,11). Likewise, homo-oligomeric assemblies of TatA have been described both in vitro (10,(12)(13)(14)(15) and in vivo (4,16,17).…”

“…Following a productive binding of a Tat substrate to TatBC, TatA is recruited (15,19,39) to initiate the transmembrane passage of the RR precursor protein. Functionally assembled Tat translocases seem to have multiple precursor-binding sites to allow for the simultaneous transport of several precursor proteins (8,40).…”