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

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Abstract

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.

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.

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