Functional properties of the sex-hormone-binding globulin (SHBG)-like domain of the anticoagulant protein S

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Abstract
Protein S (PS) possesses a sex‐hormone‐binding globulin (SHBG)‐like domain in place of the serine‐protease domain found in other vitamin K‐dependent plasma proteins. This SHBG‐like domain is able to bind a complement fraction, C4b‐binding protein (C4b‐BP). To establish whether the PS SHBG‐like domain can fold normally in the absence of other domains, and to obtain information on the specific functions of this region, we expressed the PS SHBG‐like domain alone or together with its adjacent domain EGF4. The folding of the two recombinant modules was studied by analyzing their binding to C4b‐BP. The apparent dissociation constants of this interaction indicated that both recombinant modules adopted the conformation of native PS, indicating that the PS SHBG‐like region is an independent folding unit. We also obtained the first direct evidence that the SHBG‐like domain alone is sufficient to support the interaction with C4b‐BP. In addition, both recombinant modules were able to bind Ca2+ directly, as shown by the migration shift in agarose gel electrophoresis in the presence of Ca2+, together with the results of equilibrium dialysis and the functional effect of Ca2+ on the C4b‐BP/PS interaction, confirming the presence of one Ca2+ binding site within the SHBG‐like domain. Neither recombinant module exhibited activated protein C (aPC) cofactor activity in a clotting assay, suggesting that the PS SHBG‐like region must be part of the intact molecule for it to contribute to aPC cofactor activity, possibly by constraining the different domains in a conformation that permits optimal interaction with aPC.