A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain

Abstract

Key Points• A novel FXI binding tripeptide motif has sequence Asp-PhePro (DFP).• FXI complex crystal structures reveal DFP peptides bound to the apple 2 domain.Factor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the "saucer section" of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 10 6 to 10 7 peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain. (Blood. 2016;127(23):2915-2923

Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI

“…Crystals were observed for PKa in the Morpheus screen with a reservoir containing 0.1 m carboxylic acids, 0.1 m Hepes/MOPS pH 7.5, 12.5% MPD, 12.5% PEG1000, 12.5% PEG3350 and a drop containing 0.1 m carboxylic acids, 0.1 m Tris/BICINE pH 8.5, 20% PEG 500 MME, 10% PEG 20 000. Recombinant FXI was a gift from Novo Nordisk and crystallized as described previously . Single crystals were transferred to the reservoir solution containing 20% glycerol, and flash cooled in liquid nitrogen.…”

An update on factor XI structure and function

“…Domains A1 and A4 may also contribute [82,83]. A major advance in understanding the FXI-HK interaction came with the determination of the crystal structure for FXI in complex with an eight amino acid peptide derived from the HK D6 domain called HKP [84]. The central region of D6 containing the sequence Asn-Pro-Ile-Ser-Asp-Phe-Pro-Asp (residues 583-590) binds to a pocket in the A2 domain β-sheet (Figure 6A & 6B).…”

Cell Receptor and Cofactor Interactions of the Contact Activation System and Factor XI

“…FXIIa or β-FXIIa (the isolated protease domain fragment) cleaves PK resulting in a two-chain enzyme kallikrein (PKa), consisting of a 52-kDa heavy chain and 33–36 kDa light chain corresponding to the serine protease domain and both chains are linked together by a disulfide bond. PK shares 58% amino acid sequence identity with FXI and both proteins have the characteristic feature of four apple domains (A1–A4) ( 59 , 60 ). The HK binding region on PK is localized in the central portion of the A2 domain with possible binding sites in other apple domains ( 61 ).…”

“…Activation of FXI by thrombin or α-FXIIa yields FXIa that consists of a heavy chain of four apple domains (A1-A4) and a light chain of the catalytic serine protease domain covalently linked together via a disulfide bond. Apple domains A1 and A2 contain a binding site for thrombin and HK, respectively ( 50 , 59 ). A3 has a FIX, heparin, and GPIb-IX binding site and A4 contains a cysteine residue that forms the disulfide bond needed for FXI dimer formation ( 42 ).…”