Research
Rees Group Research - Over the Decades
I have been interested in macromolecular structure for 50 years as a graduate student and faculty member, with particular emphasis on the structures and mechanisms of complex metalloproteins and integral membrane proteins involved in ATP-dependent transduction processes. The metalloprotein work was principally centered on proteins that incorporate unusual molybdenum and tungsten containing centers, including the original structure determinations defining the nitrogenase iron-molybdenum cofactor and the more widespread molybdenum cofactor that participate in many of the basic reactions of the biological nitrogen and sulfur cycles. Membrane protein studies characterized bacterial ATP-dependent transporters and mechanosensitive channels, as part of a general effort to understand how protein conformation is coupled to the cellular environment to control the movement of molecules across the membrane bilayer.
Highlights of this work follow, including references to representative reviews from our group.
Nitrogenase
The enzyme nitrogenase catalyzes the conversion of atmospheric dinitrogen to the metabolically usable form of ammonia during the process of biological nitrogen fixation. We want to understand how nitrogenase achieves this transformation under physiological conditions, which contrast sharply with the high temperatures and pressures of the industrial Haber-Bosch process. A central focus of our studies has been the structural and functional characterization of the nitrogenase component proteins (the iron (Fe-) protein and the molybdenum-iron (MoFe-) protein) and their constituent metalloclusters, particularly the iron molybdenum cofactor (FeMo-cofactor) of the MoFe-protein that provides the active site for dinitrogen reduction. We have elucidated the structure of the FeMo-cofactor and solved the first structures of a liganded form of the FeMo-cofactor with bound carbon monoxide. We subsequently generated a selenated form of the FeMo-cofactor and established that rearrangements of the cofactor can occur during turnover. Using single particle cryoEM and chemical analysis, we established from analyses of various forms of the Azotobacter vinelandii MoFe-protein that loss of homocitrate is coupled to α-subunit domain and FeMo-cofactor disordering. Under turnover conditions, the FeMo-cofactor exhibits distortions and rearrangements; these rearrangements may provide clues how the active site is activated during the catalytic cycle from a relatively stable form to a highly reactive state capable of reducing dinitrogen under ambient conditions.
ABC Transporters
ATP Binding Cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins responsible for the ATP powered membrane translocation of a wide variety of substrates. ABC transporters function as either importers, that bring nutrients and other molecules into cells, or as exporters, that pump ligands across membranes. The highly conserved ABC domains defining the superfamily provide the nucleotide-powered engine that drives transport. In contrast, the transmembrane domains creating the translocation pathway are more variable. Our group provided the first structure of an intact ABC transporter, the E. coli vitamin B12 uptake transporter BtuCD that defined the basic architecture of this transporter family. We have subsequently determined structures in multiple conformational states of ABC transporters of the E. coli MetNI methionine importer, the N. aromaticivorans Atm type transporter, and the Arabidopsis Atm3 transporter.
Mechanosensitive Channels
All organisms, from single-celled bacteria to multi-cellular animals and plants, must sense and respond to mechanical force in their external environment (shear force, gravity, touch) and in their internal environment (osmotic pressure, membrane deformation) for proper growth, development, and health. We have studied two families of prokaryotic mechanosensitive channels first identified by Kung, the mechanosensitive channel of large conductance (MscL) and of small conductance (MscS). MscL and MscS are intrinsically stretch-activated channels that open and close in response to tension applied directly to the bilayer and protect against osmotic downshock by transiently creating large pores in the membrane to release osmotic pressure gradients. Our crystallographic studies of MscL and MscS were conducted to provide structural frameworks for understanding mechanosensitive systems at the molecular level.
Photosynthetic Reaction Centers & Respiratory Complexes
Our initial efforts on membrane protein structure centered on naturally occurring membrane protein complexes relevant to photosynthesis and respiration, in collaboration with George Feher (UCSD) and Gary Cecchini (UCSF), respectively.