Spaces in Research:

We are interested in understanding the mechanisms of ion transport across biomembranes. In particular, we study heavy metal (Cu+, Cu2+, Zn2+, Co2+) transport P1B-type ATPases, a subgroup of P-ATPases. Because of their metal specificity and the chemical similarities among transition metals, these pumps also transport alternative non-physiological substrates (Cd2+, Pb2+, Ag+, Au+). P1B-ATPases share a common catalytic mechanism with the archetypical P-ATPases (Na+,K+-ATPase, Ca2+-ATPase, H+-ATPase). This mechanism couples the catalytic phosphorylation of Asp in the invariant DKTGT sequence to the transmembrane translocation of the metals. P1B-ATPases have structural features such as metal-binding signature sequences in their transmembrane segments (TM), topological arrangements with eight or six TMs, and regulatory cytoplasmic metal binding domains (MDB), that distinguish them. Consistent with their substrate specificities and direction of transport, i.e., metal efflux from the cytoplasm, they confer metal tolerance to archaea and bacteria, while in higher eukaryotes they are responsible for metal micronutrient absorption, distribution and clearance. Members of this protein family are responsible for Wilson and Menkes disease in humans.

Structure-Function Studies:

P1B-type ATPases contain 6-8 transmembrane fragments carrying signature sequences in segments flanking the large ATP binding cytoplasmic loop. These sequences made possible the differentiation of several P1B-ATPase subgroups with distinct metal selectivity: P1B-1: Cu+, P1B-2: Zn2+, P1B-3: Cu2+, P1B-4: Co2+, etc. In our structural/functional studies, we use model archaeal and prokaryote enzymes. These can be expressed in E. coli, solubilized and purified in a functional form. Using a combination of mutagenesis and functional assays we have established the stoichiometry and the metal coordination during transport for Cu+-ATPases, and the role of cytoplasmic metal binding domains. In addition, we have decribed the mechanism of metal transfer from soluble Cu+-chaperones to the transmembrane transport sites. In collaboration with Amy Rosenzweig and her group (Northwestern Univ). , we have recently solved the structure of various cytoplasmic domains. The overall structure of those involved in enzyme phosphorylation (P-domain), nucleotide binding (N-domain) and energy transduction (A-domain), appears similar to those described for the SERCA Ca2+-ATPase. However, they show different features that are characteristic of P1B-ATPases. Futures studies within this project are directed to better understand the binding of various metals to transport sites and the structure of the transmembrane region.

Biochemistry of Microbial Heavy Metal ATPases:

At the core of microbial-host interactions (symbiotic or pathogenic) is the distribution of metals and electrolytes. For instance, macrophages reduce the availability of essential Mn2+ and Fe2+ in the phagosome, while increasing K+ levels and reducing the pH. The animal and plant hosts will also generate damaging reactive oxygen species (ROS) and nitrogen species (RNS). Microrganisms respond to these secreting a number of metalloproteins (SOD, catalase, etc.) Consequently, the microbial proteins involved in the influx/efflux, storage and utilization of transition metal (Fe2+/3+, Cu+/2+, Zn2+, Mn2+, etc.), protons, ammonium, and alkali cations (K+, Mg2+, etc.) are likely to have determinant roles. Our laboratory has recently started to study the putative role of microbial heavy metal transport ATPases in the bacterial virulence.

Biochemistry of Plant Heavy Metal ATPases:

Metal micronutrients such as cooper, zinc, iron, etc. are key for plant growth and survival. However, the molecular mechanisms of metal absorption, distribution and accumulation in plants are not fully understood. It is clear that a better knowledge of these processes is needed for understanding and manipulating: a) plant nutrition; b) content of micronutrients required in the human diet; c) plant resistance to toxic heavy metals; and d) plant sequestration of heavy metals for phytoremediation. Among the molecules that transport heavy metals in plants, P1B-ATPases play a key role. Genomic information from Arabidopsis thaliana shows the presence of eight genes for heavy metal transport ATPases. We recently showed the role of HMA2 an Arabidopsis thaliana Zn-ATPase. The enzyme drives the efflux of metal (Zn and Cd) from the cell cytoplasm. hma2 knockouts show high Zn content suggesting a key role of the enzyme driving the efflux of metal from tissues. Future studies in this area include the functional characterization of other metal transport ATPases present in plants.