The First Structure of a Glycoside Hydrolase Family 61 Member, Cel61B from Hypocrea jecorina, at 1.6 Å Resolution Saeid Karkehabadi 1 , Henrik Hansson 1 , Steve Kim 2 , Kathleen Piens 3 , Colin Mitchinson 2 and Mats Sandgren 1 ⁎ 1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, P.O. Box 590, SE-751 24 Uppsala, Sweden 2 Genencor—A Danisco Division, 925 Page Mill Road, Palo Alto, CA 94304, USA 3 Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Received 1 April 2008; received in revised form 5 August 2008; accepted 7 August 2008 Available online 13 August 2008 The glycoside hydrolase (GH) family 61 is a long-recognized, but still re- condite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate- binding module and is a single-domain protein that folds into a twisted β- sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved struc- ture. In the Cel61B structure, there is no easily identifiable carbohydrate- binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61. © 2008 Elsevier Ltd. All rights reserved. Edited by M. Guss Keywords: protein crystal structure; glycoside hydrolase family 61; Hypocrea jecorina; Trichoderma reesei; Cel61B Introduction The filamentous fungus Hypocrea jecorina (pre- viously called Trichoderma reesei) 1 secretes large quantities of carbohydrate-degrading enzymes that act synergistically to degrade cellulose and related biomass components. The cellulolytic and hemicel- lulolytic machinery of this organism has been studied intensively as a model system, with a recent focus on the use in conversion of lignocellulose biomass feed stocks. These enzymes are classified in different glycoside hydrolase (GH) families in accordance with the classification system of Henris- sat and coworkers, 2,3 based on sequence similarities and consequent conservation of fold and stereoche- mical outcome of the catalyzed reaction, i.e., inver- sion (single displacement) or retention (double displacement) of the anomeric configuration. 4,5 The gene products of H. jecorina include at least five endoglucanases (EG, EC 3.2.1.4), Cel5A, Cel7B, Cel12A, Cel45A and Cel61A 6 (previously known as *Corresponding author. E-mail address: mats@xray.bmc.uu.se. Abbreviations used: CBM, carbohydrate-binding module; GH, glycoside hydrolase; NAG, N-acetyl glucosamine; NCS, noncrystallographic symmetry; PIXE, particle-induced X-ray emission; PEG, polyethylene glycol; SAD, single-wavelength anomalous dispersion. doi:10.1016/j.jmb.2008.08.016 J. Mol. Biol. (2008) 383, 144–154 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.