Molecular Basis of Heme hexacoordination in Globins

LAIA JULIO; NICOLAS VILLAGRAN DOS SANTOS; IGNACIO BORON; URIEL N. MORZAN; ALEJANDRO D. NADRA; MARCELO A. MARTI; DARIO A. ESTRIN; LUCIANA CAPECE

Chascomus

Conferencia; IV Latin American Meeting on Biological Inorganic Chemistry - V WOQUIBIO; 2014

INTRODUCTION The globin superfamily is formed by a widely distributed group of heme proteins, which are responsible for a variety of biological functions in all kingdoms of life. Many of these functions involve the coordination of small ligands to the prosthetic heme group, a process that is tightly controlled. The iron is coordinated to the four equatorial porphyrin nitrogen atoms and axially coordinated by the absolutely conserved proximal histidine F8 (HisF8), leading to the characteristic pentacoordinated (5c) state. The vacant coordination at the distal site permits the binding of the diatomic ligand and allows the protein to fulfill its function. In several globins, however, the distal site is blocked by the coordination of an endogenous residue, usually the distal histidine (HisE7), thus resulting in a bis-histidyl hexacoordinated (6c) species. This process introduces an additional mechanism to control the function of globins, as has been noticed for neuroglobin (Ngb)1, cytoglobin2, non-symbiotic plant hemoglobins3, and truncated hemoglobins4. It is important to recall that while globins are generally classified as penta or hexacoordinated, binding of exogenous ligands is only possible to the reactive 5c form. For hexacoordinated hemoglobins this is made possible thanks to the precisely regulated equilibrium between 6c and 5c states. In this scenario, understanding the molecular basis that underlies the regulation of the 5c⇆6c equilibrium is crucial for deciphering a fundamental regulatory mechanism of the physiological function of globins. EXPERIMENTAL METHODS We have applied Molecular Dynamics simulation methods to the study de 5c⇆6c equilibrium. Initial structures for the simulations were taken from the Protein Data Bank (PDBids 1D8U, 1OJ6, 1VXD, 4L2M-4MAX, and 1BIN for Rice Hemoglobin, Neuroglobin, Myoglobin, truncated hemoglobin of Synechococcus sp and Soybean Leghemoglobin, respectively). All strutcures were minimized and thermalized. Then, 100 to 200 ns (depending on the case) MD simulations were performed, for the 5c and 6c structures. All simulations were performed with the PMEMD module of the Amber12 Package. 5 Visual Molecular Dynamics (VMD)6 and the Ptraj Module of Amber12 was used to analyze the obtained trajectories. Essential dynamics analysis was applied in order to determine the principal and low frequency movements of the proteins along the MD simulations. RESULTS AND DISCUSSION In this work we applied state-of-the-art molecular dynamics simulation techniques to a selection of examples of 5c and 6c globins, in which we analyze the key structural and dynamical determinants of the heme coordination state. These include: mammalian proteins as myoglobin (Mb) and neuroglobin (Ngb), plant globins such as Rice Hemoglobin and soybean leghemoglobin, and bacterial hemoglobins as the truncated hemoglobin of Synechococcus sp (GlbN)7. These examples allowed us to identify key regions in the protein structure for the occurrence of the 5c⇆6c equilibrium. In particular, the main differences between Ngb and in 6c plant hemoglobins and 5c globins has shown to be concentrated in the structure and flexibility of the CD region. 8 The importance of this region was confirmed experimentally in Ngb by means of chimeric proteins. 9 In the case of Rice Hemoglobin, we have observed that dimmerization of the protein modifies the 5c⇆6c equilibrium by affecting the dynamics of the CD region.10 Additionally, the analysis in bacterial truncated hemoglobin of Synechococcus sp indicated that the EF region plays an important role in the transition, which is in this case the most flexible protein region. In this case, the transition involves the movement of the E helix assisted by a conformational change of the EF region, while the CD region remains in a similar position for both coordination states. CONCLUSION The results of this work highlight the importance of the flexible regions in the process and provide a detailed molecular understanding of the structural and dynamical differences betweeen 6c and 5c globins.