Three-dimensional Solution Structure and Conformational Plasticity of the N-terminal Scavenger Receptor Cysteine-rich Domain of Human CD5

• Published in: Journal of molecular biology Vol. 378; no. 1; pp. 129 - 144
• Main Authors: Garza-Garcia, Acely, Esposito, Diego, Rieping, Wolfgang, Harris, Richard, Briggs, Cherry, Brown, Marion H., Driscoll, Paul C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 18-04-2008
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; CD5 Antigens - chemistry; CD5 Antigens - genetics; CD5 Antigens - immunology; CD5 lymphocyte antigen; Cysteine - chemistry; Escherichia coli; Escherichia coli - genetics; Humans; inferential structure determination; Molecular Sequence Data; NMR spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Pichia - genetics; Pichia pastoris; protein structure; Protein Structure, Tertiary - genetics; Receptors, Scavenger - chemistry; Receptors, Scavenger - genetics; Receptors, Scavenger - immunology; scavenger receptor cysteine-rich domain; Solubility; Solutions; Temperature; NOE; inferential structure determination; SRCR; RMSD; TOCSY; CD5d1; HEK; mAb; eCD5d1m; M2BP; protein structure; scavenger receptor cysteine-rich domain; MARCO; SPR; MD-SA; GdmHCl; ISD; HSQC; NMR spectroscopy; CD5 lymphocyte antigen; pCD5d1
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.02.006

The lymphocyte receptor CD5 influences cell activation by modifying the strength of the intracellular response initiated by antigen engagement. Regulation through CD5 involves the interaction of one or more of its three scavenger receptor cysteine-rich domains present in the extracellular region. Here, we present the 3D solution structure of a non-glycosylated double mutant of the N-terminal domain of human CD5 expressed in Escherichia coli (eCD5d1m), which has enhanced solubility compared to the non-glycosylated wild-type (eCD5d1). In common with a glycosylated form expressed in Pichia pastoris, the [15N,1H]-correlation spectra of both eCD5d1 and eCD5d1m exhibit non-uniform temperature-dependent signal intensities, indicating extensive conformational fluctuations on the micro–millisecond timescale. Although approximately one half of the signals expected for the domain are absent at 298 K, essentially complete resonance assignments and a solution structure could be obtained at 318 K. Because of the sparse nature of the experimental restraint data and the potentially important contribution of conformational exchange to the nuclear Overhauser effect peak intensity, we applied inferential structure determination to calculate the eCD5d1m structure. The inferential structure determination ensemble has similar features to that obtained by traditional simulated annealing methods, but displays superior definition and structural quality. The eCD5d1m structure is similar to other members of the scavenger receptor cysteine-rich superfamily, but the position of the lone α helix differs due to interactions with the unique N-terminal region of the domain. The availability of an experimentally tractable form of CD5d1, together with its 3D structure, provides new tools for further investigation of its function within intact CD5.