Molecular Dynamics Simulation of Leishmania major Surface Metalloprotease GP63 (Leishmanolysin) Gianluca Bianchini, 1 Alessio Bocedi, 1,2 Paolo Ascenzi, 2,3 Enrico Gavuzzo, 4 Fernando Mazza, 1, * and Massimiliano Aschi 1, * 1 Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita ´ L’Aquila,L’Aquila, Italy 2 Istituto Nazionale per le Malattie Infettive I.R.C.C.S. Lazzaro Spallanzani, Roma, Italy 3 Dipartimento di Biologia e Laboratorio Interdipartimentale di Microscopia Elettronica, Universita ´ Roma Tre, Roma, Italy 4 Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Roma, Italy ABSTRACT One of the molecular factors con- tributing to Leishmania sp. virulence and pathogen- esis is the major surface metalloprotease GP63, alternatively called leishmanolysin, MSP, and PSP (EC 3.4.24.36). Here, the molecular dynamics simula- tion of Leishmania major GP63 in water at pH 7 is reported. Upon solvation, GP63 undergoes a sharp structural relaxation with respect to the crystal structure. The fluctuation pattern occurs essen- tially in solvent-exposed nonstructured regions. By contrast, the active site turns out to be rigid. Essen- tial dynamics and dynamic-domain analyses, both carried out on the equilibrated portion of GP63, show that the fingerprint fluctuations of GP63 are practically characterized by the motion of a large part of the N-terminal domain. These results appear to be in line with substrate recognition and (pro)en- zyme activation played by the N-terminal domain of GP63. A systematic analysis among a series of 10 homologs of GP63 also shows that the residues involved in the interdomain bending result highly conserved. This finding also suggests possible rela- tionship between the maintainance of proteolytic activity and the similarity of the dynamical proper- ties of the related enzymes. Proteins 2006;64:385–390. © 2006 Wiley-Liss, Inc. Key words: Leishmania major; metalloprotease; mo- lecular dynamics; essential dynamics; dynamic domain INTRODUCTION Leishmania are parasitic trypanosomatids which cause a broad spectrum of diseases, affecting about 12 millions of people in tropical and temperate zones of both the Old and New Worlds. Leishmaniasis can be fatal (visceral form), grossly disfiguring (mucocutaneous form), or relatively mild, being localized and in some cases even self-healing (some forms of cutaneous leishmaniasis). 1–3 Aspartic-, cysteine-, serine-, and metalloproteases ap- pear to be relevant to several aspects of the Leishmania sp. life cycle and to affect parasite– host relationships. Among others, Leishmania sp. proteases play a key role in the penetration of the parasite into host cells, participate in the nutrition of the parasite at the expense of the host, and are involved in the escape mechanisms of the parasite from the host’s immune system. Therefore, proteases are regarded as promising targets in the therapeutic treat- ment of leishmaniasis. In fact, protease inhibitors block Leishmania sp. replication and differentiation, providing an alternative to traditional therapy against drug- resistant parasites. Furthermore, Leishmania sp. pro- teases are attractive vaccine candidates. 4 –12 GP63, alternatively called leishmanolysin, MSP, and PSP (EC 3.4.24.36), is a metalloprotease belonging to the metzincin class, representing the major surface protease of Leishmania sp. GP63 contributes to parasite virulence and pathogenesis, participating in resistance of promastigotes to complement-mediated lysis and in receptor-mediated uptake of leishmania. In addition to the surface-associated enzyme, recent evidence suggests that active GP63 is released from promastigotes by autoproteolysis of GPI- linked GP63 and/or secretion of intracellular GP63. Inter- estingly, GP63 is a drug target and vaccine candi- date. 7,13,14 GP63 is expressed on both promastigotes and amasti- gotes of all Leishmania sp. and has a wide substrate specificity and pH optimum, in particular GP63 from L. major is optimally active at neutral to alkaline pH. GP63 degrades many protein substrates including casein, azoca- sein, gelatin, albumin, hemoglobin, fibrinogen, and CD4 molecules on human T cells. 7 GP63 (478 amino acid residues) contains predominantly -sheet secondary structure and consists of the N- terminal, the central, and the C-terminal domains. The N-terminal domain contains the His264 and His268 cata- lytic residues within the HisGluXxxXxxHis signature. Abbreviations: DynDom, dynamic domain; ED, essential dynamics; GP63, Leishmania major surface metalloprotease (also named leish- manolysin or MSP or PSP); MD, molecular dynamics; MMP-8, human neutrophyl collagenase; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation. Grant sponsor: Ministero dell’Istruzione, dell’Universita ´ e della Ricerca of Italy; Grant number: PRIN-COFIN-2003. *Correspondence to: Massimiliano Aschi, Fernando Mazza Diparti- mento di Chimica, Ingegneria Chimica e Materiali, Universita ´ L’Aquila, Via Vetoio, Coppito, 67100 L’Aquila, Italy. E-mail: aschi@caspur.it or mazza@univaq.it Received 24 September 2005; Accepted 3 November 2005 Published online 17 May 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21009 PROTEINS: Structure, Function, and Bioinformatics 64:385–390 (2006) © 2006 WILEY-LISS, INC.