Tinkering with coordination in globins

LUCÍA CHISARI, IGNACIO BORON, LUCIANA CAPECE, MARCELO A. MARTI, DARIO A. ESTRIN AND ALEJANDRO D NADRA

Rosario

Congreso; IV Congreso Argentino de Bioinformática y Biología Computacional; 2013

Asociación Argentina de Bioinformática y Biología Computacional

Background Hemeproteins are ubiquitous proteins capable of carrying out multiple functions such as: acting as enzymes, performing redox reactions and oxygen transport, and sensing small molecules (O2, NO and CO), among others. In particular, myoglobin is a pentacoordinated hemeprotein that allows oxygen transport inside the muscle. Recently, another member of this family has been found: neuroglobin. Neuroglobin is present in vertebrates and other organisms´ nervous system. It binds small gaseous ligands in the heme group´s sixth coordination position. In absence of these ligands, the heme group binds to an endogenous histidine. It has been suggested that this bond and the equilibrium between the hexacoordinated and the pentacoordinated state regulate ligand affinity and provide the protein with major thermal stability. Recent work by Nadra et al. [1] has revealed that neuroglobin´s changes in coordination correlate to changes in the C-CD-D region. To achieve a detailed analysis of this situation, two chimeras were constructed by swapping neuroglobin´s and myoglobin´s CD loops (both in silico and in vitro). Molecular dynamics and spectroscopic results suggest that each chimeric protein shifted its coordination state according to its CD region, as shown for the partially hexacoordinated myoglobin or the subtly pentacoordinated Ngb. Goal With the aim of further displacing the coordination equilibrium, we designed point mutations to enhance the observed behaviour. Results By exploring the contacts stabilizing the CD region itself and the interactions between this region and the rest of the protein, we identified several candidate residues to be mutated. For some of them we performed molecular dynamics simulations to confirm its relevance in protein dynamics. We have found a contact between a lysine and a glutamic acid in myoglobin which is absent in neuroglobin (in the position equivalent to myoglobin´s lysine we find an alanine). This contact may account for myoglobin´s preference for the pentacoordinated state. Therefore, we have made an additional mutation on the chimeras in silico: we have changed myoglobin´s lysine for an alanine and neuroglobin´s alanine for a lysine. We have run molecular dynamics simulations of these proteins with encouraging results and we will be generating the recombinant proteins.