Mutational Alterations in the Homotetrameric Chaperone SecB That Implicate the Structure as Dimer of Dimers

Abstract

Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys 76 , Val 78 , and Gln 80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.SecB is a chaperone from Escherichia coli involved in the facilitation of export of proteins from the cytoplasm to the outer membrane, which lies beyond the cytoplasmic membrane, and to the periplasm, the aqueous space between the two membranes. Like all chaperones it binds its ligands by virtue of their non-native state. The binding is highly selective for unstructured proteins; however, no motif among the ligands has been found that might serve as a recognition element for SecB (1, 2). Mutant strains of E. coli that produce altered species of SecB and are defective in protein export have been isolated by Kumamoto and co-workers (3). Analyses of complexes between SecB and a ligand, precursor maltose-binding protein, isolated by co-immunoprecipitation from these mutant strains led to the proposal that the aminoacyl residues in the region from 74 to 80 at the even numbered positions, i.e. Phe had no effect on the efficiency of co-immunoprecipitation of SecB and ligand; thus, it seems that the effect on export observed in vivo is the result of a defective interaction with other components of the export pathway. Here we have further investigated the nature of the defects exhibited by the altered species of SecB and conclude that the region implicated in binding of the precursors is crucial in maintaining the quaternary structure of SecB. SecB is a tetramer of identical subunits, but the species with substitutions at the positions 76, 78, and 80 exist as dimers. We conclude that these residues must be involved either directly or indirectly in forming the interface of dimers that normally associate to form tetramers and that substitutions at these positions shift the equilibrium so that the tetramer dissociates. EXPERIMENTAL PROCEDURESProtein Purification-Mature galactose-binding protein and precursor galactose-binding protein were purified as described (4, 5). SecB was purified from strain BL21(DE3) pJW25 (6). SecBL75Q, SecBE77K, and SecBF74I were purified from s...

SecB, a molecular chaperone with two faces

“…2). The quaternary organization agrees well with the observed dynamic dimer-tetramer equilibrium of SecB in solution 9 .…”

“…These residues are located on a surface-exposed β-strand, but they point towards the core of the molecule and are not part of the peptide-binding groove 8 . In these mutants, which show only mild translocation defects 13 , the tetramer-dimer equilibrium in solution is shifted towards the dimer 9 . As these residues do not directly contribute to the dimer-dimer interface, it is conceivable that they indirectly disrupt this interface and thereby impair preprotein binding.…”

“…Specific mutations in E. coli SecB (C76Y, V78F and Q80R) disrupt the interaction between SecB and preproteins 9,13 . These residues are located on a surface-exposed β-strand, but they point towards the core of the molecule and are not part of the peptide-binding groove 8 .…”

“…Even with a structure for the ternary SecB-SecA-preprotein complex, this question might be difficult to answer as the transfer reaction requires the highaffinity interaction between SecA and SecB that is observed only with the SecYEG-bound SecA. It is of interest to note that some of the mutations that interfere with SecA binding 19 and the tetramer-dimer equilibrium 9 map at different faces of a β-strand 13 that contacts the long α-helices constituting the dimer-dimer interface 8 . Hypothetically, high-affinity binding of SecA to SecB could cause a reduction in the preprotein binding affinity by influencing the SecB dimer-dimer interfacial contact in analogy to the mutations that shift the tetramer-dimer equilibrium.…”

“…When fused to a bacterial Tat signal sequence, LacZ is not translocated across the inner membrane 9 . This is probably because the signal peptide-LacZ fusion rapidly forms a very large homotetramer immediately following translation.…”