Current Research

Douglas Rees is interested in the structure and function of metalloproteins and membrane proteins, particularly those involved in ATP-dependent transduction processes.

Our research focuses on how dimeric ATPases in multi-subunit assemblies direct unidirectional reactions in two systems: ATP-dependent electron transfer in nitrogenase catalyzed nitrogen fixation and ATP-dependent membrane transport by ATP Binding Cassette (ABC) transporters. While these are superficially disparate processes, "under the hood," these two systems exhibit significant mechanistic and structural similarities that we are exploring in our research.

Nitrogenase and Nitrogen Fixation (this work was supported in part by GM045162)
Nitrogenase, the enzyme solely responsible for replenishing the nitrogen cycle, catalyzes the ATP-dependent reduction of N2 to ammonia. The mechanistic questions related to how nitrogenase overcomes the kinetic stability of the NN triple bond have intrigued chemists for the past 100 years. Nitrogenase consists of two oxygen-sensitive metalloproteins, the molybdenum iron (MoFe-) protein and the iron (Fe-) protein. The MoFe-protein contains the active site for substrate reduction, while the Fe-protein, which binds and hydrolyzes ATP, is the only known electron donor that supports dinitrogen reduction. The MoFe-protein coordinates two copies each of two unusual metalloclusters designated the FeMo-cofactor and the P-cluster. Functionally, the FeMo-cofactor represents the site of substrate reduction, while the P-cluster receives electrons from the Fe-protein [4Fe:4S] cluster.

An important characteristic of the nitrogenase reaction is that substrates only bind to forms of the MoFe-protein reduced by 2-4 electrons relative to the “as-isolated” form; efficient generation of these states requires turnover with reduced Fe-protein and MgATP. Consequently, high-resolution structural methods require trapping of catalytically relevant species generated under these conditions. We obtained crystals of CO inhibited MoFe-protein after incubating the nitrogenase proteins under turnover conditions in the presence of CO, followed by concentrating and crystallizing the MoFe-protein in ~4 hrs under conditions minimizing CO dissociation. The structure revealed a CO molecule bound to the FeMo-cofactor bridging two irons and replacing a belt-sulfur atom (S2B). The inhibited protein may be reactivated by incubation under turnover conditions in the absence of CO and is accompanied by the reappearance of S2B.

The finding that S2B can be reversibly substituted with CO raises the possibility of exchanging exogenous species into this site. We have recently developed a method to achieve catalysis dependent, site-selective incorporation of selenium into FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The crystal structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, the same position identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and was used as the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures, the exchangeability of all three belt-sulfur sites of FeMo-cofactor was demonstrated, providing direct insights into unforeseen rearrangements of the FeMo-cofactor during catalysis.

ABC Transporters
A major component of our interest in nitrogenase reflects the general implications of this system for nucleotide-dependent transduction processes. As a counterpoint, we have also been intrigued by ABC transporters that use ATP to achieve the unidirectional translocation of ligands across membranes by an alternating access mechanism. In the conformation competent for ATP hydrolysis, nucleotides are sandwiched between conserved sequence motifs from the two ABC domains, and the accompanying rearrangements are transmitted to the transmembrane domains. While there have been impressive structural advances, including multiple intermediates reported for the maltose and vitamin B12 importers by Chen and Locher, respectively, the connection to kinetics remains largely unrealized. We wish to make these connections between structure, kinetics and mechanism for two ABC transporters, the NaAtm1 exporter of glutathione derivatives and the MetNI methionine importer.

NaAtm1: an exporter of glutathione derivatives 
Members of the Atm1/ABCB7/HMT1/ABCB6 family of ABC exporters have been implicated in cellular iron-sulfur cluster biosynthesis and detoxification processes, likely involving glutathione (GSH) complexes. Through crystallographic and functional analyses, we established that an Atm1-type ABC exporter from the bacteria Novosphingobium aromaticivorans (NaAtm1) binds glutathione derivatives and confers protection against silver and mercury toxicity when expressed in E. coli. NaAtm1 represents the first ABC exporter to have the binding site for specific ligands defined at high resolution. Two binding sites have been identified in the inward-facing conformation of NaAtm1 for oxidized GSSG near the site of mutations linked to sideroblastic anemia, and are further suggestive of a binding mode for a tetra-GSH coordinated [2Fe:2S] cluster. Comparison of NaAtm1 to previously solved ABC exporters indicates that this family has an articulated construction, where rigid elements are connected by flexible joints with key ligand interactions positioned at the boundaries between conserved structural elements. As a consequence of this architecture, ligand binding is linked to the conformational changes underlying the ATPase-coupled substrate translocation.

MetNI: a methionine importer 
Escherichia coli MetNI mediates the high affinity uptake of D- and L-methionine. Early in vivo studies by Kadner established that the uptake of external methionine is repressed by the level of the internal methionine pool, a regulatory phenomenon termed transinhibition. We have determined the crystal structure of MetNI in the inward facing conformation. To address the transinhibition mechanism, we studied the kinetics of ATP hydrolysis using detergent solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by L-methionine. Significantly, the underlying mechanism - ligand binding to regulatory domains prevents association of the nucleotide binding subunits, effectively "turning off the engine" by inhibiting ATP hydrolysis - exemplifies a general mechanism for regulating the activity of ATP-dependent transduction systems at the protein level.

As of March 10, 2016

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