Research Highlight #118
Conformational Changes in MBP During Maltose Transport Through the MalFGK2 Complex
Amy L. Davidson1, Mariana Austermuhle1, and Candice S. Klug2
1Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas; 2Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin
The class of proteins termed ATP-binding cassette (ABC) transporters is one of the largest found in nature. Their capacity to move a variety of substances across a membrane using energy from the cell is fundamentally important to bacterial physiology and multidrug resistance, in addition to an array of human pathologies such as cystic fibrosis.
Transport of nutrients into bacterial cells relies on binding-protein-dependent transport systems that belong to the ABC transporter family. The most well-characterized transport system thus far in bacteria is the maltose transport system of E. coli and is therefore a well-suited model for the study of ABC transporters. The maltose transport system consists of a maltose binding protein (MBP) that is responsible for transport of maltose through the periplasm, two inner membrane proteins (MalF/MalG) that are likely alpha-helical in structure, and two copies of a cytoplasmic ATPase (MalK). It is known that MBP undergoes a conformational change upon binding maltose and then delivers it to the periplasmic side of the MalFGK2 complex by binding to MalFG. This docking triggers a conformational change in MalFG when ATP is bound to MalK2, resulting in release of maltose into the membrane complex. Upon hydrolysis of ATP to ADP by MalK2, maltose is transported through the complex and into the cell. Previously, the detailed mechanism of each intermediate in the transport process was unknown. In this study, single and double cysteines were introduced into MBP, spin labeled, and analyzed through each step of the transport process by EPR methodology. It was determined by EPR that the two sites, 41 and 211, chosen on opposite sides of a cleft, are greater than 25Å apart in the open form of MBP, which is in agreement with the crystal structure. After binding maltose, they come within about 8-10Å of each other, as the cleft closes down on the ligand. The open and closed states of MBP during the transport process were followed through both distance measurements and changes in the EPR spectra in order to determine the mechanism of transport.
It was determined that when added to the transporter in the absence of ATP, maltose-bound MBP does bind to the transporter, as evidenced by a conformational change in the one of the sites examined, but it remains in the closed, maltose-bound conformation. In order to trap the complex in the ATP bound state prior to hydrolysis, ATP and EDTA were added. A significant conformational change occurred at both sites and the protein went back to the open state, having delivered its maltose into the transporter but remained tightly bound in the open state. By adding MgCl2 into the system, allowing ATP to be hydrolyzed to ADP, the complex resumes its resting configuration and MBP is released to bind another maltose. Also, in order to trap the intermediate states after hydrolysis where ADP and Pi are both still bound, NaVi was added and resulted in an open MBP tightly bound to the complex, whereas once the Pi dissociates and only ADP is still bound, the complex resumes its resting state and MBP is released to bind another maltose. By studying specific sites on a single protein through each step of the transport process, a detailed understanding of the conformations of both MBP and the transporter complex were established for the first time. Using the maltose transport system as a model system, a general mechanism for how ATP couples transport of a number of solutes can be established that will lead to an enhanced understanding of the molecular basis for the diseases ABC transporters cause and to propose novel therapies to overcome these disorders.
Research Highlight #117
Copper Coordination in the Full-Length, Recombinant Prion Protein: A Significant Contribution from S-band EPR
Colin S. Burns1, Eliah Aronoff-Spencer2, Giuseppe Legname3, Stanley B. Prusiner3, William E. Antholine4, Gary J. Gerfen2, Jack Peisach2, and Glenn L. Millhauser1
1Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, 2Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, 3Institute for Neurodegenerative Diseases and Departments of Neurology and of Biochemistry and Biophysics, University of California, San Francisco, California 94143, and 4Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. The HGGGW segment constitutes the fundamental binding unit in the octarepeat domain (Burns et al.  Biochemistry 41, 3991; Aronoff-Spencer et al.  Biochemistry 39, 13760). Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP was investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X- and S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the thre-onine. The copper affinity is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies provide a profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.
The X-band spectra of PrP(90-101) and GGGTH both revealed superhyperfine splittings indicative of coupling to more than a single nitrogen atom. In addition to the nitrogen atoms of histidine imidazoles, deprotonated amide nitrogens may also participate in Cu2+ coordination. To determine which additional nitrogen atoms beyond that from the imidazole bind Cu2+, both PrP(90-101) and GGGTH, plus a series of sequentially labeled 15N-labeled analogues of GGGTH, were examined by S-band EPR. The improvement in spectral resolution for S-band over X-band EPR arises from a partial cancellation of g-strain- and A-strain-induced inhomogeneous broadening for specific hyperfine lines. At S-band, this cancellation selectively narrows the mI = -1/2 hyperfine line. Since 15N has a spin I = 1/2, a change in the multiplet pattern is observed if that nitrogen coordinates copper.
Spectra for PrP(90-101) and GGGTH are equivalent, each showing a splitting pattern consisting of eight or more resolved lines, and consistent with the ESEEM and X-band spectra, suggest that each construct coordinates copper in an equivalent fashion. Inspection of spectra from the 15N-labeled peptides reveals an isotope influence only for the third glycine amide, demonstrating that its amide nitrogen directly coordinates Cu2+. The S-band spectra also reveal an even number of superhyperfine lines (except for GGGTH). If the multiplet was due solely to equivalent 14N interactions, one should observe an odd multiplet pattern. An even multiplet can arise from inequivalent 14N superhyperfine interactions or an additional I = 1/2 spin, possibly from a nearby proton. In most cases, there is only small variance in the magnitude of hyperfine couplings for directly coordinated nitrogen atoms. Alternatively, there is good precedent for proton coupling to metal centers. Since all S-band spectra were obtained in D2O solution, an 1H coupling likely arises from a nonexchangeable proton within the peptide.