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How does the structure of metal transport membrane ATPases determine their functional characteristics?

The study of metal transport P1B-ATPases has been the core of our laboratory since its beginnings. Employing as a model Archaeoglobus fulgidus CopA, a thermophilic Cu+-ATPase, we established the catalytic mechanisms, transport stoichiometry, structure and function of soluble domains, and the structure of metal transport sites in the transmembrane region of these enzymes. We showed the specificity and the determining structures in distinct subfamilies of Zn2+, Cu2+, Ni2+, Mn2+ and Fe2+ ATPases. Moving forward, we attempt to understand the chemical interactions and resulting forces that drive the movement of transition metal along dehydrated protein permeation paths.

How do bacteria distribute metals to correct target enzymes while remaining tolerant to metal toxicity?

Metalloenzymes carry out reactions essential for a plethora of cellular processes. Their mismetallation has deleterious effects. In consequence, a primary cellular requirement is to target cognate metals to the corresponding targets. On the other hand, free metals compete for bona fide protein binding sites, while can also interact with numerous adventitious sites. Free redox active metals generate harmful free radicals, with the associated fitness cost These place metal sensing, chaperoning, storage, and transmembrane transport at the forefront of cell chemistry and biology. We hypothesize that these mechanisms are integrated in system wide distribution networks. Supporting this, we determined that homologous ATPases participate in branching the allocation of cytoplasmic Cu+ to distinct periplasmic destinations. We also observed that transported Cu+ is delivered to transmembrane ATPases by cognate Cu+-chaperones. In turn, these translocate the ion into accepting periplasmic chaperones. We aim now to understand the architecture and dynamics of the distribution networks. Perhaps more challenging, is our goal describing the chemical interactions determining how metals are transfer from one network element to the next while ensuring specificity.

How do bacteria metal homeostasis mechanisms determine survival in the challenging phagosomal environment of eukaryote hosts?

Mechanisms of innate immunity include higher Zn2+ and Cu+ levels and an oxidative burst in the phagosome that leads to Fe-S center disruption with a consequent high cytoplasmic Fe2+ in the bacteria. We have observed that various distinct transition metal ATPases are present in pathogenic organisms; for instance the unique Fe2+ ATPases and the Mn2+ ATPase in Mycobacterium tuberculosis. We hypothesize that pathogenic organisms have a more diverse arrange of metal transporters and homeostatic mechanism participating not just in metal tolerance but also in coping with the oxidative bust by metallation of critical enzymes.

On going projects

Coper homeostasis in the opportunistic pathogen Pseudomonas aeruginosa

Our goal is to define the mechanisms of Cu+ homeostasis in the pathogen Pseudomonas aeruginosa. This organism is an important and frequent cause of hospital acquired infections, especially in immune compromised patients. Free Cu+ is highly reactive and deleterious to cells and consequently copper tolerance systems contribute significantly to bacterial virulence.

On the other hand, it is a micronutrient required as a redox co-factor in the catalytic center of enzymes. It is not known how cellular components interact and participate in ion distribution to achieve Cu+ targeting to essential cuproenzymes and tolerance to high Cu+ concentrations. We hypothesize that cells have two Cu+ sensing/distribution networks. One is responsible for targeting Cu+ to cuproproteins and responds to changes in cuproenzymes functionality when challenged by environmental stressors.The other network maintains a cellular Cu+ quota and responds to cytoplasmic Cu+ levels. We aim to define and model these Cu+ distribution networks by using transcriptomics and metalloproteomics approaches. In addition, we are characterizing the specificity and routes of Cu+ entrance, distribution in the cytoplasm, transport to the periplasm, and final targeting to efflux systems or cuproenzymes. To this end, compartmental fluxes are determined in combination with in vivo biochemical equilibria amongst Cu+-sensing and distributing molecules. We are integrating the obtained parameters into mechanistically driven mathematical models. Departing from reductionist approaches, the project shifts the analysis of heavy metal homeostasis by considering the full range of involved elements, the biochemical equilibria in which they participate, and the integrated system response to environmental challenges.

This project is performed in collaboration with Reinhard Laubenbacher. Center for Quantitative Medicine. UConn Health.