WILLIAM A. CRAMER

Structure-Function of Membrane Proteins

Studies focus on the structure-function of integral membrane proteins, the class of proteins embedded in the membrane lipid matrix.  The functions of these proteins include photosynthetic electron transport, membrane-based energy transduction, and protein/toxin import, and outer membrane receptor function in gram-negative bacteria.  The experimental approaches are mostly biophysical (crystallography, spectroscopy, electrophysiology, liposomes), but are critically dependent upon the special nuances of membrane protein biochemistry. Topics of present interest concern the detailed trans-membrane pathway of proton transfer in the cytochrome b6f complex whose resolution is now 2.7 A, the role of lipids within the complex, superoxide production, the structure basis of trans-membrane signaling and activation of the LHC kinase in the complex, and dielectric heterogeneity in the complex.  In addition to the alpha-helical cytochrome complex, 5 structures of the OmpF (resolution 1.6-2.0 Å) and BtuB E. coli outer membrane proteins have been solved which provide insight into the mechanism of cellular import of colicin cytotoxins.

I. Function of Integral Membrane Proteins, Cytochrome b₆f Complex

Fig 1. Subunit structure and lipid binding sites in the cytochrome b₆f complex (A) Dimeric cytochrome b₆f complex from Mastigocladus laminosus (PDB accession 2E74). Color code: Cytochrome f/PetA (yellow), cytochrome b₆/PetB (cyan), Rieske Fe₂S₂ protein/PetC (orange), subunit IV/PetD (purple), PetG (green), PetL (brown), PetM (green), PetN (purple).  (B) Top view of b₆f complex along membrane normal showing 26 trans-membrane helices, the 2-fold symmetry axis, and positions of lipids and bound detergents (UDM). 

Although there are more than 50,000 independent structures of soluble proteins, approximately 300 such structures of integral membrane proteins in the Protein Data Bank, there are only about 2 dozen structures of hetero-oligomeric oligomeric membrane protein structures that have been solved to a resolution ≤ 3.0 Å.  Although these membrane proteins are of great interest in health and disease-related studies, most of these hetero-oligomeric structures are of protein complexes that function in membrane energy transduction.  A high resolution (2.7 Å) structure has been obtained of one of the three major hetero-oligomeric membrane protein complexes, the cytochrome b₆f complex, the electron and proton-transferring complex that connects the two reaction center complexes in photosynthetic electron transport. The dimeric 220-kDa complex contains 8 polypeptide subunits (Science, 2003; Proc. Natl. Acad. Sci., 2006; J. Mol. Biol., 2007; J. Biol. Chem., 2009; J. Mol. Biol., 2011; Proc. Natl. Acad. Sci., 2013) 13 trans-membrane helices, 7 prosthetic groups (4 hemes, 1 FeS cluster, 1 Chl a, 1 β-carotene), and 7 bound lipids per monomer. Several structure-function aspects of this complex are novel, including the covalently bound heme c_n that is electronically linked to heme b_n and, as a protein-bound heme, is unique in not having an amino acid side chain as an axial ligand.  Crystal structures with quinone analogue inhibitors imply that heme c_n is the electron donor to plastoquinone on the electro-chemically negative side of the membrane.  The structure provides insight to the pathways of electron and proton transfer across the membrane, and the labyrinthine transfer pathways of the hydrophobic quinone and quinol across cytochrome b₆f and bc₁ complexes.

II.  The NDH-1 electron/proton transfer complex

(NADH dehydrogenase) from cyanobacteria has a unique FMN or additional reduction site and a much smaller set of iron-sulfur clusters in its electron transfer chain compared to other known NDH complexes. The complex has been purified from the thermophilic cyanobacterium T. elongatus in small amounts using a strain with a natural internal His tag (from E.-M. Aro). Fifteen subunits were initially identified by mass spectroscopy. Moreover, two additional intact masses of 4902.7 and 4710.5 Da could be assigned after re-annotation of the T. elongatus genome. NdhP and NdhQ are predicted to contain a single transmembrane helix each; homologues are apparent in other cyanobacteria.  NdhP and NdhQ are predicted to contain a single transmembrane helix each and homologues are apparent in other cyanobacteria (Novaczyk et al., Biochemistry, 2011).

III. Protein Import:The Colicin Translocon

Hypotheses for the pathway and mechanisms of import into E. coli of the cytotoxic E colicins are partly based on structures of a complex of the colicin with its outer membrane vitamin B12 receptor (BtuB), which the colicins parasitize for import. A 2.75 Å structure of the complex of the receptor-binding domain of the endoribonucleolytic colicin E3 showed its elongate 100 Å long colicin domain to be bound in an oblique mode, in which it appears to be 'fishing' for a second outer membrane receptor-translocator (Nature Struct. Biol., 10, 948-, 2003)[Fig. 2C]. A similar structure has been obtained with the receptor-binding domain of colicin E2 (Sharma et al., JBC, 2007)[Fig.2D]. Circular dichroism and electrophysiological studies imply that the colicin is first bound tightly to the extracellular surface of BtuB, is unfolded upon binding, and then uses a very abundant outer membrane protein, OmpF, for translocation (Biochemistry, 45:10199-10207, 2006; EMBO J., 27, 2171, 2008). It is shown that colicin import across the outer membrane involves formation of a 'translocon' between BtuB and OmpF.  A 3.0 Å crystal structure was obtained of a complex between the translocation domain of colicin E3 and OmpF, and 1.6 Å of OmpF alone (EMBO J., 27, 2171, 2008). A 1.95 Å structure of the BtuB vitamin B₁₂ receptor was obtained by crystallization in the lipid cubic phase (Cherezov et al., 2006). Figs. 2A, B (top/side views)

Education

B. S., Physics, Massachusetts Institute of Technology
Ph. D., Biophysics, University of Chicago;
Postdoctoral, University of California/San Diego

Teaching 

• Bioenergetics: Energy Transduction in Biological Membranes: Course Outline
• Membrane Proteins Course Outline
• Ethics: Life and Times in Academic Research Labs Course Outline

 Recent Outside Lectures

• “Transfer of electrons, Protons, and Information in the Cytochrome b₆f Lipidic Complex of Oxygenic Photosynthesis,” NIH Roadmap Meeting to Membrane Protein Technologies, University of California, San Francisco, CA, November 27-30, 2012.
• "Structure-Function of the Cytochrome b₆f Complex of Oxygenic Photosynthesis," Department of Chemistry and Biochemisty, Arizona State University, November 15, 2012.“Lipid Functions in Cytochrome bc Complexes: an unusual event in evolution,” Conference, “How bugs kill bugs, progress and challenges in bacteriocin research,” British Biochemical Society, Nottingham, England. July 16 – 18, 2012.
• "Lipid Functions in cytochrome bc complexes: an unusual event in evolution," May 13-17, 2012. Membrane Conference, Arizona State University, Phoenix, AZ.
• “The Cytochrome b₆f Complex: an Odd Lipid Effect in Evolution; An Electrostatically Constrained Electron Transport Pathway,” May 3 – 4, 2012. Royal Society Discussion on “Structure and Dynamics of the Thylakoid Membrane,” Kavi Royal Society International Centre, Buckinghamshire, UK.
• "Lipid Functions in cytochrome bc complexes: an unusual event in evolution," May 3, 2012. Royal Society Center, Chicheley Buckinghamshire, UK.
• “Oligomeric Membrane Protein Complexes;" Symposium on "Structure-Function of Hetero-Oligomeric Membrane Proteins," February 25, 2012. Bioenergetics Subgroup, Biophysical Society, San Diego, CA.
• "Impediments in Travel through Membrane Proteins: Problems in Bioenergetics and Protein Import;" February 13, 2012. Department of Biochemistry and Biophysics, University of Stockholm, Sweden.
• "Traveling Inside the Cytochrome bc Complex of Energy-Transducing Membranes," January 5, 2012. Department of Biophysics, Albert Einstein College of Medicine, Bronx, NY.
• Seminar: “Structure-Function of Cytochrome bf Complex,” December 5, 2011, the Biophysics and Computational Biology Department, University of Illinois-Champaign/Urbana.
•  “Impediments in traveling through membrane proteins: problems in bioenergetics and protein import.” March 31, 2011, Dept. of Biochemistry & Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL.
• Organization meeting of the X-ray free electron laser. January 18 – 21, 2011, University of California/Berkley, Berkley, CA.
• “Problems and Strategies in the Crystallization and Structure Solution of the Helical Cyanobacterial Cytochrome E. coli β-barrel Omps, btuB b₆f complex,” November 16-18, 2010, 3^rd annual NIH Roadmap Meeting on Membrane Protein Technologies, La Jolla, CA.
• “The cytochrome b6f complex of oxygenic photosynthesis,” September 28, 2010, Danforth Institute Symposium on Plant Structural Biology, St. Louis, MO.
• "The Q Cycle of the Cytochrome b₆f Complex in a Structural Perspective." Chair's address, Bioenergetics Symposium, August 23, 2010, XV International Photosynthesis Congress, Beijing, China.
• "The Q Cycle in a Structural Perspective." Symposium on "Molecular Basis of Photosynthetic Energy and Electron Transfer and Related Respiratory Processes" August 18-19, 2010, Institute of Advanced Studies, Nanyang Technological University, Singapore.
• "The Q Cycle of Cytochrome bc Complexes Considered in a Structural Perspective." Symposium on "Quinone and Oxygen in Energy Coupling and Catalysis" July 23, 2010, Jagiellonian University, Kraków, Poland.
•  "Labyrinths and Barrels: Inside Membrane Proteins," at the Cambridge Healthtech Institute PepTalk on Membrane Proteins, Jan. 14, 2010, San Diego, CA.
• "Towards the Structure of the Ion Channel Formed by Alpha-Synuclein," May12, 2010, M. J. Fox Foundation, New York, NY.
• "Traffic Problems in Membrane Proteins," Skirball Institute of Biomolecular Medicine, May 14, 2010,New York University School of Medicine  New York, NY.
•  "Membrane Proteins; Life in an Oily Environment." Short Course, " Membrane Proteins; The Essential Protein Engineering Summit," May 16, 2010, Cambridge HealthCare Institute,  Boston, MA.
• Keynote address: November 14, 2009, "Traveling Inside Membrane Proteins," Symposium for 50th Anniversary, Institute for Protein Research, Osaka University, Japan.
• Presentations: Feb. 28-March 4, 2009, 53^rd annual meeting of Biophysical Society, Boston, MA.
  • Bioenergetics Sub-group: "Role of lipids in the cytochrome /b/_6 /f/complex"
  • Platform session on Protein Import: "Translocation Across Membranes: Components of Outer Membrane Colicin Translocons"
  • Mini-symposium on Electron Transfer and Energy Coupling Reactions in Organelles. "Unique structure aspects of the cytochrome /b/_6 /f/complex"

Awards/Honors

National Science Foundation Postdoctoral Fellowship, Univ. Calif/San Diego 1965-67;

NIH(NIGMS) Research Career Development Award, 1970-75;

Senior Fellow, European Molecular Biology Org., Univ. of Amsterdam, The Netherlands, 1974-75;

McCoy Award, Purdue University, Achievements in Science, 1988;

Plenary Lectures, International Congress of Photosynthesis, 1989, 2004;

Fellow, Alexander von Humboldt Foundation, 1992;

Fellow, John Simon Guggenheim Foundation, 1992-93;

C.F. Kettering Award, Amer. Soc. Plant Physiology, 1996;

Rosetta Briegel Barton Lecture, University of Oklahoma, 2001;

Maurice Hilleman Lecture, Montana State University, 2004;

Daniel Arnon Lecture, University of California/Berkeley, 2006;

Fellow, Biophysical Society, 2007;

Eminent Scholar Lecture, Oklahoma State University, 2008.