Functional epitopes of the B domain of fV short. Surface representation of the portion of the B domain bound to the A3 and A2 domains of fV short (Figure 2B) that contains the entire AR (residues 1493-1537) and its preceding segment preAR (residues 1458-1492) housing the hydrophobic patch 1481PLVIVG1486 important for recognition of TFPIα.26,29,33 Residues of the B domain are colored in white (A, F, I, L, M, P, V, W, and Y), cyan (D and E), and light blue (all others) to emphasize the contribution of HCs and acidic residues in the preAR and AR. Intramolecular interactions within the B domain (red ∗) include a hydrophobic coupling between L1544 and I755 near R1545, a H-bond between the side chains of R1534 and N1480 and a hydrophobic contact between I1498 and Y1518. A stronger H-bond connects R1534 to E1668 (cyan) in the A3 domain and anchors the R1534-N1480 interaction to the surface of the A3 domain. The segment I755-L1544 defines 4 HCs and a patch of acidic residues (AC). HC1 occupies the bottom of the B domain and contains 7 residues in a linear arrangement. HC1 continues with the hydrophobic patch 1481PLVIVG148629 as part of HC2 that also includes F1474, L1475, F1479, and L1487. Residues P1663, W1665, and F1666 nearby are colored in white on the surface of the A3 domain, with F1666 interacting with V1487 in HC2. The B domain continues with HC3 that ties the ascending and descending portions of the B domain through an interaction between I1498 and Y1518. The B domain terminates at R1545 with HC4, where L1544 contacts I755 in HC1. The HCs and AC of the B domain of fV short may orchestrate recognition of the C-terminal basic region of TFPIα and its synergistic inhibition of fXa with PS in the fV short/TFPIα/PS/fXa complex. In fV, the HCs and AC of the B domain likely provide the locale for intramolecular engagement of the BR in the B domain that keeps the cofactor in its inactive state. A recent model of the AR has proposed epitopes for binding the BR and the basic C-terminal end of TFPIα.56 Interestingly, the model identifies a hydrophobic core of 9 residues (I1495, I1497, F1498, V1503, I1513, V1516, Y1518, Y1522, and I1530) that may assist the acidic residues E1507, D1509, D1514, D1519, and D1520 in the binding interactions. The general organization is consistent with the features emerged from the cryo-EM structure of fV short, but several residues in the proposed epitopes of the AR56 are unlikely to contribute to binding. Specifically, (i) the 9 hydrophobic residues do not arrange in a well-defined core, with I1495, V1503, and I1530 being widely separated (up to 24 Å); (ii) the side chains of Y1518 and Y1522 are not exposed to solvent; and (iii) E1507 and D1509 are part of the 1507EDDY1510 wedge and completely buried under the A2 domain (Figure 4B).
