The counterreceptor binding site of human CD2 exhibits an extended surface patch with multiple conformations fluctuating with millisecond to microsecond motions

Abstract

We have used I5N NMR relaxation experiments to probe, for the glycosylated human CD2 adhesion domain, the overall molecular motion, as well as very fast nanosecond-picosecond (ns-ps) and slow millisecond-microsecond (ms-ps) internal motions. Using a novel analysis method that considers all residues, we obtained a correlation time for the overall motion of 9.5 + 0.3 ns. Surprisingly, we found a large contiguous patch of residues in the counterreceptor (CD58) binding site of human CD2 exhibiting slow conformational exchange motions (ms-ps). On the other hand, almost none of the residues of the CD58 binding site display fast (ns-ps) internal motions of amplitudes larger than what is seen for well-ordered regions of the structure. Residues close to the N-glycosylation site, and the first N-acetylglucosamine of the high mannose glycan are as rigid as the protein core. Residues conserved in the immunoglobulin superfamily V-set domain are generally very rigid.Keywords: glycosylated human CD2 adhesion domain; immunoglobulin superfamily V-set domain; "N NMR relaxation: reduced spectral density mapping CD2 is a transmembrane glycoprotein receptor expressed on the cell surface of T lymphocytes and natural killer cells and is important in mediating both cellular adhesion and signal transduction (reviewed in Moingeon et al., 1989a;Hahn et al., 1993). CD2 mediates adhesion by binding to its counterreceptor CD58 in humans (Selvaraj et al., 1987). CD2-mediated adhesion facilitates T cell recognition of antigens by the T cell receptor (Moingeon et al., 1989b: Koyasu et al., 1990 J ( w N ) , and J(w"), spectral density function derived at zero, nitrogen, and proton frequencies, respectively; Jeg(0), in the absence of exchange its value equals J(0); Juvg(wH,). the average of the spectral density function at the proton frequency as well as the sum and difference of the proton and nitrogen frequencies; T , , overall tumbling time; GlcNAc-I , N-acetylglucosamine. vation signals that synergize with those mediated by the TCR (Meuer et al., 1984;Bierer et al., 1988), and in vivo studies in rodents have shown that administration of anti-CD2 monoclonal antibodies effectively prolong allograft survival (Chavin et al., 1993;Qin et al., 1994). Moreover, the CD2-CD58 pathway plays a critical role in the maintenance and reversal of anergy (BoussiOtis et al., 1994), and anti-CD2 and anti-CD58 mAbs can specifically inhibit interleukin-12-induced proliferation and interferon y production by activated T cells (Gollob et al., 1995).Structural studies of both rat (Driscoll et al., 1991;Jones et al., 1992) and human CD2 Wyss et al., 1993;Bodian et al., 1994) have shown that the extracellular region of CD2 consists of a nine-stranded NH,-terminal immunoglobulin superfamily V-set domain and a seven-stranded membrane-proximal IgSF C2-set domain. The CD58 binding site of human CD2 is a highly charged surface area located on the GFCC'C" face of the NH2-tenninal adhesion domain (Arulanandam et al., 1993;Somoza et al., 1993). The affinity of the...

A space-time structure determination of human CD2 reveals the CD58-binding mode

“…Experimentally, we have observed slower motions in the s to ms range as well, in particular for the CD58-binding site (18). To rationalize motions at this slow time scale is still beyond the approach presented here.…”

“…They consisted of 50 distance restraints for hydrogen bonding pairs, 945 nuclear Overhauser restraints (860 intrapolypeptide, 46 intraglycan, and 39 polypeptideglycan), and 192 dihedral restraints (92 , 19 1 , 13 ␤-methylene, and 68 glycan). The dynamic constrains were adapted from previously performed NMR relaxation parameters of NH groups (18). Using relaxation rates, R N (N z ), R N (N x, y ), and R N (H z 3 N z ), spectral densities, J eff (0), J( N ), and J ave ( H ) were determined by using spectral (24) and RASMOL (28).…”

“…If the fitting results are not satisfactory, an extended model with a chemical exchange term R ex was applied. Of the 102 order parameters obtained from the experimental data Wyss et al (18), 66 main-chain order parameters with the lowest experimental errors were used for the refinement.…”

“…The adhesion domain of hCD2 forms a nine-stranded Ig superfamily V-set fold. The dynamics of hCD2 have been characterized with 15 N NMR relaxation experiments (18). Here, we have present a space-time description of the adhesion domain of CD2 based on the JAM approach.…”

Amplitudes and directions of internal protein motions from a JAM analysis of15N relaxation data

“…From 15 N NMR relaxation experiments of glycosylated hCD2 adhesion domain, it has been shown that a large contiguous patch of residues in the hCD58 binding site exhibits slow conformational exchange motions in the time range between microseconds and milliseconds. 29 Thus, hCD2 is an ideal model for studies of the mechanism and significance of internal motions.…”