Abstract
We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding protein, MalE. Binding of ATP to the MalK subunits increased the susceptibility of two tryptic cleavage sites in the periplasmic loops P2 of MalF and P1 of MalG, respectively. Lys(262) of MalF and Arg(73) of MalG were identified as probable cleavage sites, resulting in two N-terminal peptide fragments of 29 and 8 kDa, respectively. Trapping the complex in the transition state by vanadate further stabilized the fragments. In contrast, the tryptic cleavage profile of MalK remained largely unchanged. ATP-induced conformational changes of MalF-P2 and MalG-P1 were supported by fluorescence spectroscopy of complex variants labeled with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. The results suggest that complex variants exhibiting a binding protein-independent phenotype (MalF500) or containing a mutation that affects the "catalytic carboxylate" (MalKE159Q) reside in a transition state-like conformation. A similar conclusion was drawn for a complex containing a replacement of MalKQ140 in the signature sequence by leucine, whereas substitution of lysine for Gln(140) appears to lock the transport complex in the ground state. Together, our data provide the first evidence for conformational changes of the transmembrane subunits of an ATP-binding cassette import system upon binding of ATP.
Highlights
ATP-binding cassette (ABC)3 proteins exist in all living organisms and form one of the largest superfamilies
The ABC domains are characterized by a set of Walker A and B motifs that are involved in nucleotide binding and by the unique LSGGQ signature sequence [2]
These structures have provided insight into the interactions between the nucleotide-binding (ABC) domains (NBDs) and the transmembrane domains (TMDs). Notwithstanding these advances, the conformational changes in the NBDs induced by ATP binding/hydrolysis, and the means by which they are transmitted to the TMDs to effect substrate translocation remain largely to be elucidated
Summary
ATP-binding cassette (ABC) proteins exist in all living organisms and form one of the largest superfamilies. ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains (TMDs) that form the translocation pathway and two hydrophilic nucleotide-binding (ABC) domains (NBDs) that hydrolyze ATP In prokaryotes, these domains are mostly expressed as separate protein subunits, whereas in eukaryotes, especially in mammalian cells, they are usually fused into a single polypeptide chain. Further progress in understanding the structural organization of ABC transporters was achieved with the high resolution structures of BtuCD, mediating the uptake of vitamin B12 in Escherichia coli [11] and of MsbA, involved in the export of lipid A in Gram-negative bacteria [12,13,14] These structures have provided insight into the interactions between the NBDs and the TMDs. These structures have provided insight into the interactions between the NBDs and the TMDs Notwithstanding these advances, the conformational changes in the NBDs induced by ATP binding/hydrolysis The transporter is composed of the extracellular (periplasmic) receptor, the maltose-binding protein (MalE), and the membrane-bound complex comprising the hydrophobic subunits MalF and MalG and two copies of the ATPase (ABC) subunit, MalK [17]
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