The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-Larginyl-fVIIa (Fl-FPR-fVIIa). The location of the activesite probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserinedependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 Å assuming a random orientation of donor and acceptor dyes ( 2 ‫؍‬ 2/3; the orientational uncertainty totals ϳ10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane.When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 Å after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.

The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) (also called chaperonin containing TCP1 [CCT]) is a hetero-oligomeric complex that facilitates the proper folding of many cellular proteins. To better understand the manner in which TRiC interacts with newly translated polypeptides, we examined its association with nascent chains using a photo-cross-linking approach. To this end, a series of ribosome-bound nascent chains of defined lengths was prepared using truncated mRNAs. Photoactivatable probes were incorporated into these 35S- labeled nascent chains during translation. Upon photolysis, TRiC was cross-linked to ribosome-bound polypeptides exposing at least 50–90 amino acids outside the ribosomal exit channel, indicating that the chaperonin associates with much shorter nascent chains than indicated by previous studies. Cross-links were observed for nascent chains of the cytosolic proteins actin, luciferase, and enolase, but not to ribosome-bound preprolactin. The pattern of cross-links became more complex as the nascent chain increased in length. These results suggest a chain length–dependent increase in the number of TRiC subunits involved in the interaction that is consistent with the idea that the substrate participates in subunit-specific contacts with the chaperonin. Both ribosome isolation by centrifugation through sucrose cushions and immunoprecipitation with anti-puromycin antibodies demonstrated that the photoadducts form on ribosome-bound polypeptides. Our results indicate that TRiC/CCT associates with the translating polypeptide shortly after it emerges from the ribosome and suggest a close association between the chaperonin and the translational apparatus.

Coagulation factor VIIa (fVIIa), a soluble serine protease, exhibits full proteolytic activity only when bound to its cofactor, tissue factor (TF). Both proteins interact with membranes; TF is an integral membrane protein, while fVIIa binds reversibly to phospholipid surfaces via its Gla domain. In this study, we examine the extent to which the location of the fVIIa active site in the fVIIa⅐TF complex is determined by the fVIIa Gla domain. A fluorescein dye was covalently attached to the active site of fVIIa lacking the Gla domain (Gla domainless fVIIa, GD-fVIIa) via a tripeptide tether to yield fluorescein-D-Phe-Pro-Arg-GD-fVIIa (Fl-FPR-GD-fVIIa). The location of the active site of GD-fVIIa relative to the membrane surface was determined using fluorescence resonance energy transfer between the fluorescein dye in the active site of GD-fVIIa and octadecylrhodamine (OR) at the surface of phospholipid vesicles. As expected, no energy transfer was observed between Fl-FPR-GDfVIIa and OR in vesicles composed of phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) because the Gla domain is required for the binding of fVIIa to phospholipid. However, when Fl-FPR-GD-fVIIa was titrated with PC or PC/PS vesicles into which purified TF had been reconstituted, energy transfer was observed. Based on the dependence of fluorescence resonance energy transfer on OR density, the average distance of closest approach between fluorescein in the active site of Fl-FPR-GD-fVIIa⅐TF and OR at the vesicle surface was determined to be 78 Å ( 2 ‫؍‬ 2 ⁄3). Since this value is nearly the same as that obtained with intact Fl-FPR-fVIIa bound to TF, the presence or absence of the fVIIa Gla domain has only a small effect on the location of the active site in the fVIIa⅐TF complex. The extracellular domain of tissue factor therefore must be fairly rigid and fixed relative to the surface to position and maintain the fVIIa active site far above the membrane even in the absence of the fVIIa Gla domain.

Overall structure and function of TF-FVlla (1,18,19); it confers the ability to bind reversibly to anionic phospholipids. Next to the Gla-domain, and sometimes classified as part of it, is a short shetch of largely hydrophobic residues called the hydrophobic or aromatic stack. Next are two modules related to epidermal growth factor (EGF domains). EGFr, the more N-terminal of the two, contains a