Crystal structure of human arginase I at 1.29-Å resolution and exploration of inhibition in the immune response

doi:10.1073/pnas.0504027102

Crystal structure of human arginase I at 1.29-Å resolution and exploration of inhibition in the immune response

^*Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323; ^†Department de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Extremadura, 10071 Cáceres, Spain; and ^‡Tumor Immunology Program, Stanley S. Scott Cancer Center and Department of Pediatrics, Louisiana State University, Health Sciences Center, New Orleans, LA 70112

Edited by William N. Lipscomb, Harvard University, Cambridge, MA, and approved July 26, 2005 (received for review May 13, 2005)

Abstract

Human arginase I is a potential target for therapeutic intervention in diseases linked to compromised l-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH, K _d = 5 nM) and S-(2-boronoethyl)-l-cysteine (BEC, K _d = 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme–inhibitor complexes at 1.29- and 1.94-Å resolution determined from crystals twinned by hemihedry. The ultrahigh-resolution structure of the human arginase I–ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate–manganese coordination interactions and fully saturated hydrogen bond networks with inhibitor α-amino and α-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state for l-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group of l-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: chris{at}xtal.chem.upenn.edu.
Author contributions: L.D.C., G.S., A.M., P.C.R., A.C.O., F.C., and D.W.C. designed research, performed research, contributed new reagents/analytic tools, analyzed data, and wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: TH1/2, T-helper 1/2; ABH, 2(S)-amino-6-boronohexanoic acid; BEC, S-(2-boronoethyl)-l-cysteine.
Data deposition: The atomic coordinates and structure factors for human arginase I complexed with ABH and BEC have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2aeb and 1wva, respectively).