Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity Mutants

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nm for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nm; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nm. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nm for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the “balancing interface,” are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation. The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nm for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nm; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nm. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nm for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the “balancing interface,” are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation. ATP binding cassette transporters couple ATP hydrolysis to the transmembrane transport of a diverse range of compounds. Members of the ATP binding cassette transporter superfamily are characterized by two membrane-integral domains that each contain 6 or more membrane spanning helices, but are otherwise poorly conserved, and two peripheral ATP binding cassette domains that display sequence conservation across the entire superfamily (1Hyde S.C. Emsley P. Hartshorn M.J. Mimmack M.M. Gileadi U. Pearce S.R. Gallagher M.P. Gill D.R. Hubbard R.E. Higgins C.F. Nature. 1990; 346: 362-365Crossref PubMed Scopus (949) Google Scholar). In addition to the membrane complex, ATP binding cassette systems that catalyze nutrient uptake have primary receptors (binding proteins) that serve two functions: they provide a high affinity binding site for the transported molecule and they regulate the ATPase activity of the integral membrane complex. We are interested in the function of the primary receptors in the transport process. As a group, these proteins have been intensively studied by x-ray crystallography and other biophysical techniques (for a review, see Ref. 2Quiocho F.A. Ledvina P.S. Mol. Microbiol. 1996; 20: 17-25Crossref PubMed Scopus (450) Google Scholar). They typically contain two domains separated by a hinge region; the substrate binds in the cleft between the two domains, and the protein undergoes a large conformational change, leading to closure of the cleft. With respect to the maltose transport system, domain closure in the binding protein is thought to be the first step toward molecular shape recognition by the membrane complex (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar, 4Hor L. Shuman H.A. J. Mol. Biol. 1993; 233: 659-670Crossref PubMed Scopus (71) Google Scholar), although it has been shown that both substrate-loaded and substrate-free binding proteins have a role in the transport cycle (5Bohl E. Shuman H.A. Boos W. J. Theor. Biol. 1995; 172: 83-94Crossref PubMed Scopus (44) Google Scholar, 6Shilton B.H. Mowbray S.L. Protein Sci. 1995; 4: 1346-1355Crossref PubMed Scopus (21) Google Scholar, 7Merino G. Boos W. Shuman H. Bohl E. J. Theor. Biol. 1995; 177: 171-179Crossref PubMed Scopus (51) Google Scholar, 8Ames G.F. Liu C.E. Joshi A.K. Nikaido K. J. Biol. 1996; PubMed Scopus Google Scholar). The and of the and conformational changes in the binding protein used maltose-binding surface plasmon wild-type maltose-binding MBP with a deletion of residues and maltose-binding protein with and maltose-binding protein the mutations of both and the membrane complex of integral membrane and and the peripheral ATP binding cassette used maltose-binding surface plasmon wild-type maltose-binding MBP with a deletion of residues and maltose-binding protein with and maltose-binding protein the mutations of both and the membrane complex of integral membrane and and the peripheral ATP binding cassette have transport and of ATP hydrolysis by the role of binding protein affinity the transport was to MBP with affinity for without residues in either the maltose binding site or in thought to with structures of MBP in both the closed and open have been F.A. J. Biol. PubMed Google Scholar, F.A. 1992; PubMed Scopus Google Scholar), and they show that binding of maltose results in a large conformational of the the two domains that the substrate is the cleft. In MBP is in the open conformation B.H. M.M. Mowbray S.L. J. Mol. Biol. 1996; PubMed Scopus Google is to closure of the ligand binding cleft, either in the hinge or in the interface the ligand binding an interface the opposite of the hinge the ligand binding site to the protein in an open conformation in the closed conformation, interface is and to interface affect the and the maltose affinity of the is by a by and Biol. PubMed Scopus Google Scholar), in which they that of groups the interface or by increased the affinity of the protein for maltose. 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Biol. 1996; PubMed Scopus Google Scholar). the of the for and with the J. 1995; Scopus Google to a between the crystal structures of the closed and open of MBP F.A. J. Biol. PubMed Google Scholar, F.A. 1992; PubMed Scopus Google Scholar), and the solution of and The J. 1995; Scopus Google was used to the scattering the crystal structures of open MBP F.A. J. Biol. PubMed Google Scholar, F.A. 1992; PubMed Scopus Google and closed MBP F.A. J. Biol. PubMed Google Scholar, F.A. 1992; PubMed Scopus Google Scholar). these scattering are and the in conformation between and MBP is These scattering used to the crystal structures with the scattering for the proteins in In the presence of the conformation of MBP in solution or that of the ligand-bound conformation in the crystal F.A. J. Biol. 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The by which the mutations of ligand binding and the more the in The residues in the ligand-bound conformation, for either the wild-type or and in the the mutations affect the open conformation of the binding protein and that the mutations affect the of of the open conformation. the results are they that the of the mutations is to the of a that of the closed binding protein the binding be to between the open and closed conformations, in both the presence and absence of maltose In the KD a of the and is The maltose binding site is not in the closed conformation, and maltose is shown binding and the open conformation The of the a for the in the mutations have the toward the closed conformation by the of of the open conformation, the of MBP and the of maltose a for the by which the mutations the affinity for are two results that are not The first is the mutations have the conformational of the binding protein the open to the they have the conformation of the binding using to it be that the of binding protein is (for that the in by the mutations is not by The absence of the conformation is an it has been shown that MBP is to ATP hydrolysis by (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google and it has been that the closed conformation, or to was in the absence of the closed conformation is in it is to it cause a significant of the In are consistent with MBP in a open of and that the mutations have significant the conformational of the protein The is the in the of the As the responsible for the of is which is in the balancing interface and has interactions that are the protein changes conformation. Our structural both and in solution by that are changes by the mutations that the the mutations a and in The of structural that the in is of a in the of that the molecular of the are by the balancing interface for MBP is by a in which it was that but not undergoes large a to of a that was to of the domains K. P. J. PubMed Scopus Google Scholar). 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