Theoretical study of ligand migration in Hemeproteins: Truncated Hemoglobin N and Nitrophorin 7

Abstract

The diversity of functional roles played by heme proteins in all kingdoms of life is to a large extent regulated by the thermodynamic and kinetic properties of their interaction with small gaseous molecules.

The aim of this PhD thesis is to examine the structural and dynamical properties of two hemeproteins, the truncated hemoglobin N from M. Tuberculosis and nitrophorin 7 from R. Proxilus, and to examine their relationships with the biological role of these proteins. Particular attention has been paid to the diffusion of small ligands between the internal cavities in the two hemeproteins, and the entry/release pathways from the protein matrix to the solvent. Qualitative and semi-quantitative agreements between experiment and simulations are obtained for the identities of the cavities that physically trap the ligand and for the connections between them.

The truncated hemoglobin N (trHbN) is believed to constitute a defense mechanism of M. Tuberculosis against NO produced by the macrophages during the initial growth infection stage, which is converted to the harmless nitrate anion, through the chemical reaction NO+ FE(II)-O2 ->FE(III)+ [NO3]-. The dual path ligand-dependent mechanism proposed in previous studies of the group ensures the access of NO to the heme cavity in the oxygenated form of the protein, which should warrant survival of the microorganism under stress conditions and allowing the bacillus to stay in latency. As a consequence, the processes mediated by trHbN are worth for searching a therapeutical intervention, since the inactivation of the NO scavenging should reduce significantly the bacteria resistance.

In this work we have validated the importance of the PheE15 residue in the previous proposed dual path mechanism, as PheE15 was proposed to act as a gate that regulates the access of NO to the heme cavity in the oxygenated form of the protein. Thus, we have studied the impact of three residue mutations of the gate residue, PheE15Ala, PheE15Ile and PheE15Tyr, in the ligand migration mechanism and in the NO detoxification activity. The results support the gating role played by PheE15, because all the mutations are predicted to either block the long branch of the tunnel (PheE15Ile, PheE15Tyr) or to induce structural alterations that affect the passage of NO at the entrance of the tunnel (PheE15Ala).

After release of the NO3- anion, the protein, which rests in its ferric form, is assumed to be recycled by a putative reductase, rendering trHbN in the ferrous form suitable to bind a new O2 molecule, thus ensuring an efficient detoxification mechanism. In order to gain insight into the reduction process, we have examined the interaction between trHbN and a flavodocin reductase (FdR) from E. Coli, which has shown to be very efficient in restoring the ferrous form of the protein. Thus, our studies have yielded a 3D model of the complex between trHbN and FdR, which allows to identify the residues implicated in the binding of the two proteins. Moreover, the model predicts that the heme and flavin cofactors are close (between 6 and 9 Å) in the complex, which facilitates an efficient electron transfer, as reinforced by the calculated electron couplings. Nitrophorin 7 is a hemeprotein implicated in the NO transport, which can be found in the saliva of blood feeding insects. One of the bugs, Rhodnius prolixus, is the causative agent of Chagas disease, and is responsible for a high number of deaths (approximately 15000 each year). Among the nitrophorin family, we have examined nitrophorin 7, which presents three extra residues in N-terminal sequence and the unique ability to bind membranes negatively charged. The results point out the existence of up to three inner cavities, which may define an internal pathway for migration of a gaseous ligand (NO) from the heme to the back of the protein. This topological feature is not present in other nitrphorins, such as NP4, and justifies a more complex kinetic rebinding scheme in NP7. In conjunction with the ability to interact with membranes, these findings might support a specific role in NO-controlled release.

Both projects are carried out in closed collaboration with experimental groups, and we believe that in the future such collaborations will allow the development of new strategies with therapeutically implications in these diseases.

El proyecto a desarrollar se centrará en los aspectos dinámicos del transporte de ligandos en hemoproteínas, centrándose en dos tipos principales. Por un lado la hemoglobina truncada N de Mycobacterium tuberculosis, y por otro nitroforina.

La hemoglobina truncada N desempeña un papel muy importante como mecanismo de defensa del microorganismo, pues transforma el óxido nítrico (NO) generado por los macrófagos durante las etapas iniciales de respuesta a la infección en anión nitrato. Ello, pues, permite la subsistencia del microorganismo, que permanece en estado de latencia, dificultando la recuperación del paciente de tuberculosis. En consecuencia, dicha proteína aparece como una diana de potencial valor terapéutico, puesto que su inactivación debería reducir significativamente la resistencia del microorganismo. Los estudios del Prof. Luque han perseguido dilucidar los aspectos moleculares que subyacen en la capacidad de la proteína de captar O2 y NO, y de llevar a cabo la reacción química de conversión a nitrato. En particular, se ha propuesto un mecanismo de transporte de ligandos, y el objetivo que se plantea es su validación mediante el examen de diversos mutantes obtenidos al modificar el residuo que controla la migración a través del canal.

En cuanto a nitroforina, es una hemoproteína transportadora de NO que se halla en la saliva de insectos cuya alimentación se basa en sangre. Uno de ellos, Rhodnius prolixus, está implicado en la enfermedad de Chagas, que causa un número elevado de muertes (aproximadamente 15000 por año). De entre los diversos tipos de nitroforinas, nuestro interés se centra en nitroforina 7 (NP7), que difiere en el extremo N-terminal respecto a otras nitroforinas. El objetivo del trabajo se centra en determinar el efecto de dichas diferencias sobre la capacidad de captación y transporte de NO.

En ambos casos, los proyectos se llevan a cabo en colaboración con grupos experimentales, y confiamos que dicha colaboración permita abrir estrategias de intervención terapéutica.