Journal of Structural Biology 152 (2005) 84–89 www.elsevier.com/locate/yjsbi 1047-8477/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2005.08.002 Short communication Structure of a non-camelized human M12-V H domain at 1.5Å resolution Rajneesh Kumar Gaur ¤ Institute for Molecular Biotechnology VII, Aachen University, Worringerwegl, 52074 Aachen, Germany Received 5 May 2005; received in revised form 10 August 2005; accepted 11 August 2005 Available online 7 September 2005 Abstract A non-camelized human V H domain has been crystallized through limited in vitro proteolysis of scFvM12 antibody fragment. The protease addition results in the complete degradation of the M12-V L domain, linker, and puriWcation tags. The structure solved up to 1.5 Å resolution having good stereochemistry with a R cryst factor of 15.8% and R free factor of 19.7%. Dihedral angle values comparison of the Wrst and the second complementarity-determining region (CDR) of M12-V H domain with an average values show a signiWcant devia- tion; therefore, M12-V H domain structure indicates either the existence of a new canonical subclass or a link among the subclasses of canonical main-chain conformation in V H 3 family. The presence of uncommon hydrogen bond between Ser-H50 and Tyr-H97 has pull- ing eVect on CDR-H3 loop. The interface area buried by CDR-H3 loop indicates the partial coverage of the hydrophobic V L –V H inter- face. The isolated M12-V H domain was found soluble up to 0.35 mM. This result would be helpful in structure based designing of an isolated human single domain antibody fragments for biotechnological and pharmaceutical applications such as cancer.  2005 Elsevier Inc. All rights reserved. Keywords: Human V H ; Non-camelized; Crystal structure; Limited proteolysis 1. Introduction Antibodies are the frontline defense molecules of the vertebrate’s immune system. The smallest portion of the full size antibody that retains the binding aYnity and the speciWcity against the antigen is made up of variable domain of the heavy (V H ) 1 and the light chain (V L ) (Bedzyk et al., 1990). In contrast to the V L domain, V H domain often retains the antigen-binding speciWcity because the CDR-H3 loop is the major contributor to antigen binding (Chothia et al., 1985). The single variable domain (V H ) of the conven- tional heavy chains have been generated in the past (Ward et al., 1989), but the major impetus in this direction came after the discovery of the fully functional variable heavy chain antibodies (V H H) in Camelidae (Hamers-Casterman et al., 1993). Since then, many attempts have been made to generate either humanized or human V H domain. Because of their small size and reduced immunogenicity, isolated V H domains are of fundamental importance where enhanced biodistribution eYciency and the eVective avascular tissue penetration capability are desirable (Carter and Merchant, 1997). Human MUC1 is a polymorphic, type I transmembrane glycoprotein containing a variable number of 20 amino acid tandem repeats ‘PDTRPAPGSTAPPAHGVTSA’ (Hinoda and Imai, 1994). In adenocarcinomas, MUC1 undergo overexpression and have exposed protein core due to aberrant glycosylation (Hilkens et al., 1989), rendering the main immunodominant epitope ‘PDTRP’ accessible. The hypoglycosylated MUC1 in cancer cells is a suitable immunotarget for diagnosis and therapy. Recently, large array of murine monoclonal antibodies directed against MUC1 peptide have been raised and studied (Price et al., 1998). More often, the repetitive use of murine monoclonal * Fax: +1 317 274 4686. E-mail address: gaur@molbiotech.rwth-aachen.de. 1 Abbreviations used: CDR, complementarity-determining region; Fab, antibody fragment; Fo, observed structure factor amplitude; Fc, calculat- ed structure factor amplitude; Fv, variable fragment; HAMA, human anti- mouse antibody; HEL, hen egg lysozyme; MUC1, mucin-1; rms, root mean square; scFv, single chain variable fragment; V H , heavy chain vari- able domain; V L , light chain variable domain; V H H, variable domain of heavy chain antibody from camels and llamas.