Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency

Abstract

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 1∶1 complex in solution. The interaction is independent of sugarbinding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.MCFD2 | ERGIC-53 | calcium-binding protein | cargo receptor | intracellular lectin C ombined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII) (1, 2) Extensive genetic analysis of F5F8D patients identified two genes that are associated with this disorder. Genetic mutations in LMAN1 account for approximately 70% of F5F8D families, whereas the remaining 30% families possess mutation in MCFD2 (3, 4). LMAN1 encodes the transmembrane lectin ERGIC-53 (ER-Golgi intermediate compound protein of 53 kDa) (5), which possesses a luminal carbohydrate recognition domain (CRD) with specificity for highmannose-type oligosaccharides (6, 7) and forms dimers or hexamers stabilized by disulfide bonds formed in its stalk domain (5, 8). By contrast, the product of MCFD2 is a soluble luminal protein with two EF-hand Ca 2þ -binding motifs (9). It has been shown that MCFD2 interacts with the CRD of ERGIC-53 in a Ca 2þ -dependent manner (9-11). Accumulating evidence indicates that this complex recycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC) and thereby functions as a cargo receptor in the early secretory pathway of FV and FVIII (12).To gain a better understanding of the molecular mechanisms underlying the sorting and trafficking of these coagulation factors, it is essential to shed light on the structural basis of the cooperative interplay between ERGIC-53 and MCFD2. The crystallographic data of the CRD of rat ERGIC-53 have revealed that it com...

The COPII pathway and hematologic disease

“…68,69 LMAN1 binds these 2 clotting factors through its CRD, while MCFD2 binds them through the EF-hand domains. LMAN1 and MCFD2 also interact with each other through CRD and EF-hand motifs 70,71 ; however, the specific sites of interaction are distinct from the binding sites for FV and FVIII. LMAN1-deficient mice demonstrate decreased FV and FVIII levels (ϳ 50% of normal), and slightly distended ER with accumulation of ␣1-antitrypsin in hepatocytes, though ␣1-antitrypsin plasma levels are not reduced.…”

Causes of the Production of Multiple Forms of β-Galactosidase byBacillus circulans

“…The sedimentation velocity experiments were performed in buffer D using a Proteomelab XL-1 Analytical Ultracentrifuge (Beckman-Coulter, Fullerton, CA) by a previously reported method. 52,53) A 390-mL portion of -Gal-A at a concentration of 0.56 mM, 2.8 mM, or 5.6 mM was sedimented at 35,000 rpm at 20 C using 12-mm charcoal-filled double sector centerpieces with quartz windows, while -Gal-C (0.9 mM, 1.9 mM, and 7.4 mM) was sedimented at 40,000 rpm. Sedimentation behavior was monitored with absorbance detection optics, and all the sedimentation velocity data were analyzed by the continuous sedimentation coefficient distribution cðsÞ model included in the SEDFIT11.71 software program.…”

Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII

“…Therefore, these results imply that ␤1 is directly involved in MCFD2 binding, consistent with the crystal structures of the CRD-MCFD2 complex. 18,19 To identify critical amino acid residues in the ␤1 region that are involved in MCFD2 binding, we developed a model of the LMAN1-MCFD2 complex using molecular docking methods (detailed methods and results in supplemental Document 1) from the nuclear magnetic resonance structure of MCFD2 (PDB code: 2VRG) 33 and a homology model of the human CRD (supplemental Figure 1A), based on the crystal structure of the rat CRD (PDB code: 1R1Z). 30,31 In this model, the N-terminus of the CRD (primarily ␤1) directly contacts MCFD2 (supplemental Figure 2).…”

“…15 MCFD2 can interact with FV and FVIII independent of LMAN1 and the EF hand domains of MCFD2 are sufficient for binding both LMAN1 and FV/FVIII. 6,7 During the preparation of this manuscript, crystal structures of the CRD-MCFD2 complex were published, 18,19 which identify the binding surface consisting of the EF hand domain of MCFD2 and the N-terminal side of the CRD. Most MCFD2 missense mutations either disrupt the protein structure or change a crucial amino acid residue involved in binding the CRD.…”

“…Most MCFD2 missense mutations either disrupt the protein structure or change a crucial amino acid residue involved in binding the CRD. 18,19 LMAN1 was first reported to exist as both homodimers and homohexamers in cells, maintained by 2 membrane proximal cysteines (C466 and C475). 20,21 By using nondenaturing analysis techniques together with cross-linking studies, LMAN1 was later demonstrated to exclusively exist as homohexamers in cells.…”