Structural Characterization of a Periplasmic Lipoprotein Associated with Magnesium Homeostasis in Salmonella enterica

Abstract

The divalent magnesium cation (Mg2+) is imperative to the survival of all living organisms on earth. It stabilizes different macromolecular and cellular structures, and plays a vital role in a vast number of essential biochemical reactions. Due to its significance, many organisms have evolved complex biochemical systems to adapt to varying environmental levels of Mg2+. Additionally, the ability to adapt to environments with limiting Mg2+ is required by many bacteria to cause disease in humans. Gaining insight into how bacteria maintain homeostasis when in magnesium deficient environments will provide critical knowledge in finding new treatments for antibiotic resistant bacteria. Through a genetic screen, we have identified a novel periplasmic lipoprotein of unknown structure and biochemical function that rescues growth of a mutant strain of Salmonella enterica that is unable to respond to magnesium deficient environments. Interestingly, this lipoprotein has a single domain of unknown function that is conserved among a large number of disease causing enteric bacteria. To elucidate the structural characteristics of the protein, circular dichroism (CD) and X‐ray crystallography techniques are being used. CD experiments have revealed that the protein is composed primarily of beta‐sheet secondary structure, which confirms the secondary structure predicted by homology modeling. Thermal denaturations analyzed by CD reveal a single state reversible transition with a melting temperature of approximately 71 °C. Additionally, we are progressing towards obtaining the first high resolution atomic structure of this protein. We have developed reproducible crystal conditions for the native protein and have successfully obtained atomic resolution X‐ray diffraction data which suggests more than one monomer per asymmetric unit. Initial attempts at molecular replacement with this data have so far been unsuccessful. Selenomethionine (SeMet) was incorporated into the native protein, and has also been successfully crystallized. Obtaining X‐ray diffraction data on these crystals will provide the phasing information necessary to solve the first atomic resolution structure of this protein. The structure will ultimately provide us with knowledge of this protein's role in magnesium homeostasis within Salmonella.Support or Funding InformationThis work was supported by the University of Wisconsin‐ La Crosse Undergraduate Research and Creativity grant (DR), McNair Scholars program (DR), and a University of Wisconsin‐ La Crosse Faculty Research grant (JM). The authors would also like to thank Professor James Keck (University Wisconsin, Department of Biomolecular Chemistry) for allowing access to the Advanced Photon Source. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006).