Role of C-terminal segment in DC-SIGN multivalent binding. The CRDs are linked to the tetramerization coiledcoil neck domain with some degree of flexibility in both DC-SIGN and DC-SIGNR. Interestingly, a short C-terminal segment of 16-amino acid long is found in the CRD/neck junction region in DC-SIGN, but it is absent in DC-SIGNR. Therefore, we reason that this segment may act as a steric wedge to maintain the upright CRD orientation and multivalent binding properties observed for DC-SIGN, but not DC-SIGNR, and that its removal may affect DC-SIGN's multivalent binding property. To investigate this possibility, a truncated DC-SIGN with the C-terminal segment removed (named as DC-SIGN-C) was constructed and confirmed by HRMS (SI Fig. ). DC-SIGN-C was found to form stable tetramers with roughly the same hydrodynamic sizes as DC-SIGN and DC-SIGNR (e.g. Dh = ~14, ~15 and ~15 nm for DC-SIGN, DC-SIGNR and DC-SIGN-C, respectively; SI Fig. ). As with DC-SIGN, the Q274 residue of DC-SIGN-C was also mutated to a cysteine for site-specific labeling of Atto-594, giving a labeling efficiency of ~75% according to the HRMS (SI, Fig. ).