Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Joshua Haynes - Fisk University
Co-Author(s): Darlene Martin, Rukiayah Warner, and Steven Damo, Fisk University, TN
Transition metals serve as essential enzymatic or structural cofactors in biological systems. It has even been estimated that up to 1/3 of the proteome requires a transition metal for proper function. Despite their importance, our fundamental understanding of metalloproteins is still in its infancy. For example, it is not possible to predict specific metal binding sites of a protein based on sequence alone. The ultimate goal of our work is to establish a rationale for the specificity of metal binding sites in proteins. Calprotectin (CP) is a member of the S100 class of the EF-hand calcium binding protein family comprised of the heterodimer of S100A8 and S100A9 subunits. CP has two distinct metal binding sites, each located at opposite ends of the dimer interface. Site 1 can bind either Zinc (Zn 2+) or Manganese (Mn 2+) with high affinity. Site 2 can only bind Zn 2+. Our goal is to understand the molecular determinants of metal specificity in Sites 1 and 2 of CP and engineer a CP mutant that is selective for Mn2+ at Site 1. According to the Irving-Williams series, proteins will have higher affinity for Zn2+ than Mn2+ .However, Mn2+ has higher affinity for oxygen ligands than nitrogen ligands. We hypothesize that it is possible to mutate the residues in Site 1 in order to create a CP mutant that is selective for Mn2+. Using site directed mutagenesis, we mutated one of the six histidine coordinate residues of CP and measured its affinity for Mn2+ and Zn2+ using isothermal titration calorimetry. Interestingly, the kd for Mn2+ is 148 nM and the kd for Zn2+ is 379 nM, which suggests the CP mutant has a higher affinity for Mn2+. Moving forward, we will be using x-ray crystallography and EPR spectroscopy to characterize this mutant and inform future mutagenesis experiments.
References: Yannone, Steven M., Sophia Hartung, Angeli L. Menon, Michael Ww Adams, and John A. Tainer. ‘Metals in Biology: Defining Metalloproteomes.’ Current Opinion in Biotechnology 23.1 (2012): 89-95. Damo, S. M., T. E. Kehl-Fie, N. Sugitani, M. E. Holt, S. Rathi, W. J. Murphy, Y. Zhang, C. Betz, L. Hench, G. Fritz, E. P. Skaar, and W. J. Chazin. ‘Molecular Basis for Manganese Sequestration by Calprotectin and Roles in the Innate Immune Response to Invading Bacterial Pathogens.’ Proceedings of the National Academy of Sciences 110.10 (2013): 3841-846.Not Submitted
Funder Acknowledgement(s): We gratefully acknowledge the National Science Foundation for funding these studies. Joshua Haynes is supported by the TSLAMP Bridges to the Doctorate program, NSF HRD1500320. This work is also supported in part by a NSF/HBCUP RIA HRD1400969 awarded to Steven Damo.
Faculty Advisor: Steven Damo, firstname.lastname@example.org