Journal of Molecular Biology
Three-dimensional Solution Structure and Conformational Plasticity of the N-terminal Scavenger Receptor Cysteine-rich Domain of Human CD5
Introduction
CD5 is a transmembrane protein expressed constitutively on the surface of T cells and a subset of B cells. Its expression can be upregulated on all B cells by a variety of stimuli.1 CD5 is generally recognised as a modulator of lymphocyte activation. The hyper-reactive phenotype of CD5-negative cells2, 3 and other data4, 5, 6 suggests that the primary role of CD5 is to dampen the signal of the antigen receptor, thereby raising the threshold for leukocyte response. However, reports indicate that CD5 is also able to act as an enhancer of leukocyte function in T cells7, 8, 9, 10 and thymocytes.11
Biochemical and functional data suggest that CD5 mediates cell–cell communication through its extracellular region.12, 13, 14, 15, 16, 17 Soluble recombinant constructs of the CD5 ectodomain (CD5ext) have been shown to be able to bind a receptor and induce a response in B cells,12, 13 and to ameliorate experimental autoimmune diseases in vivo.12, 18 That the effect of CD5ext in vivo was found to be species-specific in one study12 and not in another18 highlights the need to delineate the binding properties of CD5 at the atomic level.
The extracellular region of CD5 contains three domains (CD5d1, CD5d2 and CD5d3) that belong to the scavenger receptor cysteine-rich (SRCR) superfamily. CD5d1 and CD5d2 are separated by a 25 residue proline- and threonine-rich region, and both possess N-linked glycans; CD5d2 and CD5d3 are directly linked in tandem.19 Blocking studies of CD5 monoclonal antibodies (mAbs) provide evidence that the N-terminal domain of CD5 is involved in ligand binding.13, 20, 21
SRCR domains have a globular disulphide-bonded α/β fold of around 110 residues and are found mainly in secreted and/or membrane-anchored metazoan proteins. The number of cysteine residues in an SRCR domain varies from four to eight. In SRCR domains with eight cysteines, the disulphide bond connectivity is (in order of occurrence of the cysteines in the polypeptide sequence) C1–C4, C2–C7, C3–C8 and C5–C6.22, 23, 24 Every known SRCR domain has at least two putative intradomain disulphide bonds, one of which is always equivalent to C3–C8. Traditionally, the SRCR superfamily had been divided in two groups, SRCR A and SRCR B, according to the number and position of the cysteines present.25 A more robust classification based on genomic organisation has since been proposed, in which SRCR A domains are those encoded by two exons and SRCR B members are those encoded by a single exon.26, 27
Earlier, we reported attempts to obtain human CD5d1 in recombinant form for analysis of its structure and function.23, 28 Recombinant N-glycosylated CD5d1 can be obtained in significant yield from Chinese hamster ovary (CHO) cells and Pichia pastoris yeast cultures, but these materials have proved refractory to structure determination. Here we report the production of CD5d1 expressed in Escherichia coli (eCD5d1). This non-glycosylated version of CD5d1 displays nuclear magnetic resonance (NMR) spectroscopic characteristics similar to those of the glycosylated forms, but has poor solubility. To obtain a 3D solution structure, we developed a double mutant form of eCD5d1 that was soluble at the concentration required for a complete NMR study. In common with the N-glycosylated forms of this domain, the NMR spectra of eCD5d1 are characterised by non-uniform temperature-dependent peak intensities, suggesting an underlying dynamic plasticity. We describe the application of inferential structure determination (ISD),29 an unbiased methodology to calculate structures from sparse and noisy NMR data, to eCD5d1. Finally, the resulting structure is compared to other members of the SRCR superfamily.
Section snippets
Production and solubility enhancement of CD5d1 expressed in bacteria
The eCD5d1 construct comprises an N-terminal His6 tag, a thrombin cleavage site, and human CD5 residues Arg25 to Glu134 (numbering according to UniProt entry P06127); Arg25 represents the N-terminal residue of native, cell surface CD5 following cleavage of the signal peptide. In BL21(DE3) Gold cells, eCD5d1 is expressed in high yield as insoluble aggregates. The aggregates were solubilised using 6 M guanidinium hydrochloride and then folded in vitro by dilution into a pH 9.0 buffer with
A structurally tractable but conformationally dynamic form of CD5d1
By engineering apolar-to-polar residue substitutions, we obtained a highly soluble double mutant of CD5d1, V88D/V97K eCD5d1 (eCD5d1m), which is amenable to NMR-based solution structure determination. Evaluation of the effect of the substitutions on the reactivity of recombinant CD5d1 with mAbs recognizing native CD5 and comparison of the NMR spectra of the N-glycosylated wild-type pCD5d1 and the unglycosylated double mutant demonstrated that eCD5d1m retains the overall structure of native
Cloning, expression, purification and in vitro folding
The DNA sequence comprising residues 25–134 of hCD5 was amplified by PCR from a mammalian expression construct,23 incorporating a restriction site sequence for NdeI at the 5′ end, and a stop codon followed by an XhoI recognition sequence at the 3′ end. The PCR product was then digested and ligated into the pET15b vector (Novagen). E. coli BL21(DE3) Gold (Stratagene) cells were transformed with the construct and then grown in LB medium with 50μg/ml of carbenicillin at 37 °C with shaking for 8 h.
Acknowledgements
Aspects of this work were supported by the MRC (to P.C.D., D.E. and A.G.G.), BBSRC (to R.H. and P.C.D.), EMBO (to W.R.), and the Mexican National Council for Science and Technology, CONACyT (to A.G.G.). W.R. thanks Ernest Laue for providing the infrastructure for the ISD work and Michael Nilges for providing the Lognormal extensions for CNS. We thank Neil Barclay and Fred Smith for insightful comments.
References (66)
- et al.
The Ca2+/calmodulin-dependent kinase type IV is involved in the CD5-mediated signaling pathway in human T lymphocytes
J. Biol. Chem.
(1997) - et al.
Native soluble CD5 delivers a costimulatory signal to resting human B lymphocytes
Cell. Immunol.
(1996) - et al.
Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain
J. Biol. Chem.
(1996) - et al.
Crystal structure of the third extracellular domain of CD5 reveals the fold of a group B scavenger cysteine-rich receptor domain
J. Biol. Chem.
(2007) - et al.
The SRCR superfamily: a family reminiscent of the Ig superfamily
Trends Biochem. Sci.
(1994) - et al.
CD6-ligand interactions: a paradigm for SRCR domain function?
Immunol. Today
(1997) - et al.
When monomers are preferred: a strategy for the identification and disruption of weakly oligomerized proteins
Structure
(2005) - et al.
Structural characterisation of the human alpha-lactalbumin molten globule at high temperature
J. Mol. Biol.
(2003) - et al.
Glycoproteins: glycan presentation and protein-fold stability
Structure
(1999) - et al.
Determinants of backbone dynamics in native BPTI: Cooperative influence of the 14–38 disulfide and the Tyr35 side-chain
J. Mol. Biol.
(1998)
Backbone dynamics of the EGF-like domain of heregulin-alpha
J. Mol. Biol.
Aromatic rings act as hydrogen bond acceptors
J. Mol. Biol.
The structure of the extracellular region of human hepsin reveals a serine protease domain and a novel scavenger receptor cysteine-rich (SRCR) domain
Structure
Crystal structure of the cysteine-rich domain of scavenger receptor MARCO reveals the presence of a basic and an acidic cluster that both contribute to ligand recognition
J. Biol. Chem.
Protein production by auto-induction in high density shaking cultures
Protein Expr. Purif.
ANSIG — a program for the assignment of protein H-1 2D-NMR spectra by interactive computer-graphics
J. Magn. Reson.
The solution structure of the domain from MeCP2 that binds to methylated DNA
J. Mol. Biol.
T-Coffee: a novel method for fast and accurate multiple sequence alignment
J. Mol. Biol.
MMTSB Tool Set: enhanced sampling and multiscale modeling methods for applications in structural biology
J. Mol. Graph. Model.
CD5 expression by B lymphocytes and its regulation upon Epstein-Barr virus transformation
Proc. Natl Acad. Sci. USA
Lymphocyte populations and immune responses in CD5-deficient mice
Eur. J. Immunol.
A role for CD5 in TCR-mediated signal transduction and thymocyte selection
Science
Origins and functions of B-1 cells with notes on the role of CD5
Annu. Rev. Immunol.
CD5 signal transduction: positive or negative modulation of antigen receptor signaling
Crit. Rev. Immunol.
CD5, an important regulator of lymphocyte selection and immune tolerance
Immunol. Res.
Simultaneous ligation of CD5 and CD28 on resting T lymphocytes induces T cell activation in the absence of T cell receptor/CD3 occupancy
J. Immunol.
Bispecific antibody-mediated target cell-specific costimulation of resting T cells via CD5 and CD28
Cancer Res.
The cytoplasmic domain of CD5 mediates both TCR/CD3-dependent and -independent diacylglycerol production
J. Immunol.
CD5 costimulation up-regulates the signaling to extracellular signal-regulated kinase activation in CD4+CD8+ thymocytes and supports their differentiation to the CD4 lineage
J. Immunol.
Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes
J. Expt. Med.
A role for CD5 in cognate interactions between T cells and B cells, and identification of a novel ligand for CD5
Int. Immunol.
The identification and characterization of a ligand for bovine CD5
J. Immunol.
CD5 is a potential selecting ligand for B cell surface immunoglobulin framework region sequences
J. Expt. Med.
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