Stoichiometry of the large conductance bacterial mechanosensitive channel of E-coli. - A biochemical study

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent cr ystallographic study of the MscL homologue from M. tuberculosis reveals a p entameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches, Detergent puri fied 6His-MscL in solution and MscL in the membrane could be chemically cro sslinked with the products displaying ladderlike patterns on SDS gels, Thre e crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product, DSS produced additi onal bands corresponding to larger complexes although the pentamer band app eared to be the predominant product at high levels of crosslinker. It is no t clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of mul tiple species. Thus the longer spacer arm could permit both intra- and inte rcomplex linkages. Nonetheless, the patterns obtained with all agents are c onsistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subu nit tandems yields low numbers of functional channels as compared to expres sed monomers, The double-tandem assemblies must have an even number of subu nits and crosslinking in the membrane confirmed hexamerization. Gel chromat ography clearly demonstrated that the channels formed from the double tande ms were larger; than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tande ms form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the poly peptide precursor. The channel is thus a pentameric core with the 'extra' s ubunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thu s, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.