The key structural role of Arg266 in human Cystathionine ​ β ­synthase

LAIA JULIO; DARIO A. ESTRIN; LUCIANA CAPECE

Rio de Janeiro

Congreso; V Latin American Protein Society Meeting; 2016

Cystathionine β-synthase (CBS) is an unusual enzyme that requires the cofactors heme and pyridoxal-5?phosphate (PLP) to catalyze the condensation of homocysteine and serine to give cystathionine. This transsulfuration reaction represents one of two major metabolic avenues for clearing homocysteine. Malfunctions of this enzyme are associated to a wide range of pathologies. Human CBS (hCBS) displays a dimeric structure, and each subunit is organized into three structural domains (Figure 1A): i) the N-terminal domain which contents a heme-binding site, ii) the central catalytic core, which displays the fold of the type II family PLP-dependent enzymes and iii) the C-terminal region, where the allosteric cofactor S-adenosylmethionine (AdoMet) binds.Interestingly, and in opposite to most heme proteins, the heme group in this protein is not involved directly in the enzymatic reaction, and its function still unknown. Even though a distance of 20 Å separates the heme from the active site, it has been observed that changes in the redox and coordination states of the iron provoke a diminution of activity. The iron in the heme group of CBS is hexacoordinated in the ferric state, displaying a cysteine and a histidine residue bound in the two axial coordination sites.The heme group is connected with the catalytic site through a portion of alpha-helix (Figure 1B). R266 is located in this helix and interacts with the coordinated cysteine. Mutations on R266 were reported to affect the enzyme activity.In this work we study the role of the heme group in hCBS by means of extensive classical molecular dynamics (MD) simulations, using the Amber14 package. We have run MD simulations of hCBS for the ferric and ferrous states in both the hexacoordinated and pentacoordinated states. We have analyzed the interactions between the heme ligands and the protein, in order to understand the structural connection between the heme site and the catalysis. We specifically explore the role of R266 by means of in-silico generated R266 mutants. Our results indicate that the lost of the interaction between Cys52 and Arg266 induces a structural change in the position of the heme group, highlighting the key role of Arg266 in hCBS function, consistently with the reported site-directed mutagenesis studies. Furthermore, the results presented here provide a possible structural and dynamical role of the heme in the regulation of the enzymatic activity in hCBS.