Crystal Structures of a Psychrophilic Metalloprotease Reveal New Insights into Catalysis by Cold-Adapted Proteases Nushin Aghajari, 1 * Filip Van Petegem, 2 Vincent Villeret, 2 Jean-Pierre Chessa, 3 Charles Gerday, 3 Richard Haser, 1 and Jozef Van Beeumen 2 * 1 Institut de Biologie et Chimie des Prote ´ines, UMR 5086, Laboratoire de Bio-Cristallographie, CNRS et Universite ´ Claude Bernard Lyon I, Lyon, France 2 Laboratorium voor Eiwitbiochemie en Eiwitengineering, Universiteit Gent, Gent, Belgium 3 Laboratoire de Biochimie, Institut de Chimie B6, Universite ´ de Lie `ge, Lie `ge, Belgium ABSTRACT Enzymes from psychrophilic or- ganisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomo- nas species (PAP), solved to 2.1- and 1.96-Å resolu- tion, respectively. Comparative studies of PAP struc- tures with mesophilic counterparts show that the overall structures are similar but that the conforma- tion of the substrate-free active site in PAP re- sembles that of the substrate-bound region of the mesophilic homolog, with both an active-site ty- rosine and a substrate-binding loop displaying a conformation as in the substrate-bound form of the mesophilic proteases. Further, a region in the cata- lytic domain of PAP undergoes a conformational change with a loop movement as large as 13 Å, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions. Proteins 2003;50:636 – 647. © 2003 Wiley-Liss, Inc. Key words: extremophile; psychrophile; crystallog- raphy; adaptation; temperature INTRODUCTION Numerous bacteria live at temperatures close to 0°C. To survive and grow at such conditions, these organisms have evolved several adaptations of their cellular components, especially of their enzymes. 1–4 The cold-adapted proteins have an increased turnover number and physiological efficiency (k cat /K m ) at low and moderate temperatures. The structures of four psychrophilic enzymes have recently been determined by crystallography, 5–9 while in the past homology modelling was more commonly used. 10 –14 Com- parison with their mesophilic counterparts has allowed us to pinpoint to some extent the molecular basis of cold adaptation. In general, it is accepted that cold-adapted proteins are more flexible, with a reduced number of stabilizing interactions. 1–4 Loops are often longer and more hydrophilic, 4 the proline and arginine contents can be lower, and there is often an increased number of glycine residues. The surface of the molecular structures can display a lower content of charged residues and a higher proportion of hydrophobic side-chains. 5,14 The number of known psychrophilic enzyme structures is still small, however, and more studies are required to improve the understanding of the structural basis of cold adaptation of these enzymes. Moreover, for comparative studies it is critical to have the structures of mesophilic (and, if avail- able, thermophilic and hyperthermophilic) counterparts to account for changes that may be linked to psychrophily. Here, we report the structures of a psychrophilic alka- line protease (PAP) from Pseudomonas TAC II 18 sp. in two different crystal forms. The organism was isolated from Antarctica and the protein characterized. 15 The 463-residue enzyme is 3 times more active at 20°C than a mesophilic counterpart from Pseudomonas aeruginosa. At 45°C, the psychrophilic enzyme is rapidly inactivated and the protein is sensitive to small concentrations (2 mM) of ethylenediaminetetraacetic acid (EDTA). At present, two mesophilic homologs with known tertiary structure have been characterized. One of them, the alkaline protease from P. aeruginosa (AP, 470 residues) shows 66% sequence identity with PAP and its structure was determined with and without a tetrapeptide in the active site. 16,17 Another metalloprotease, serralysin, also known as Serratia pro- Grant sponsor: Fund for Scientific Research-Flanders; Grant num- ber: G.0068.96; Grant sponsor: Bijzonder Onderzoeksfonds (Univer- sity of Ghent); Grant number: GOA 12050198; Grant sponsor: E.U.; Grant numbers: BI04-CT96-0051 and CT97-0131; Grant sponsor: Centre National de la Recherche Scientifique; Grant sponsor: Fonds National de la Recherche Scientifique; Grant numbers: 2.4523.97 and 2.4515.00; Grant sponsor: Re ´gion Wallonne; Grant number: 9613492. Both Nushin Aghajari and Filip Van Petegem contributed equally to this work. *Correspondence to: Jozef Van Beeumen, Laboratorium voor Eiwit- biochemie en Eiwitengineering, Universiteit Gent, B-9000 Gent, Bel- gium. E-mail: jozef.vanbeeumen@rug.ac.be or Nushin Aghajari, Insti- tut de Biologie et Chimie des Prote ´ines, UMR 5086, Laboratoire de Bio-Cristallographie, CNRS et Universite ´ Claude Bernard Lyon I, F-69367 Lyon, Cedex 07, France. E-mail: n.aghajari@ibcp.fr Received 20 November 2001; Accepted 1 August 2002 PROTEINS: Structure, Function, and Genetics 50:636 – 647 (2003) © 2003 WILEY-LISS, INC.