Human α2-macroglobulin structure Location of Cys-949 residues within a half-molecule measured by fluorescence energy transfer

AbstractTo localize the pair of thiol-ester-derived cysteine-949 residues within a half-molecule of α2M and estimate the internal diameter of this α2-macroglobulin trap, we have measured the separation between these cysteines. We unexpectedly found that the two cysteines of intermediate form α2M had different reactivity, which permitted selective modification of one cysteine with dansyl, and the other with fluorescein fluorophores. From fluorescence energy transfer measurements, we calculated a separation of 41 ± 10 Å between these fluorophores. This indicates that the Cys-949 residues are probably located at the perimeter of the trap with an internal diameter at least as large as this separation

Thiol ester role in correct folding and conformation of human α2-macroglobulin Properties of recombinant C949S variant

AbstractTo determine the role of the thiol ester in the folding of human α2-macroglobulin (α2M) in the active conformation, we have characterized a recombinant variant of α2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S α2M behaves like methylamine-treated plasma α2M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric α2M does not require a pre-formed thiol ester. Active α2M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S α2M followed by thiol ester formation and a conformational change that gives the native active state

Specificity and reactive loop length requirements for crmA inhibition of serine proteases

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (~102–104 M−1sec−1), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (kdiss ~10−7 sec−1), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (kcat/KM of ~106 M−1 sec−1). N-terminal sequencing confirmed that the P1 Arg–P1′Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1′Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases ~10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (105–106 M−1sec−1), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1′Cys–P2′Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites