The telomerase ribonucleoprotein complex ensures complete replication of eukaryotic chromosomes. Telomerase RNA, TER, provides the template for replicating the G-rich strand of telomeric DNA, provides an anchor site for telomerase-associated proteins, and participates in catalysis through several incompletely characterized mechanisms. A major impediment towards understanding its non-templating roles is the absence of high content structural information for TER within the telomerase complex. Here, we used selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine the structure of Tetrahymena TER free in solution and bound to tTERT in the minimal telomerase RNP. We discovered a striking difference in the two conformations and established direct evidence for base pair triples in the tTER pseudoknot. We then used SHAPE data, previously published FRET data, and biochemical inference to model the structure of tTER using discrete molecular dynamics simulations. The resulting tTER structure was docked with a homology model of tTERT to characterize the conformational changes of tTER that attend binding to tTERT. Free in solution, tTER appears to contain four pairing regions: stems I, II, and IV, which are present in the commonly accepted structure, and stem III, a large paired region that encompasses the template and pseudoknot domains. Our interpretation of the data and subsequent modeling affords a molecular model for telomerase assemblage in which a large stem III of tTER unwinds to allow proper association of the template with the tTERT active site and formation of the pseudoknot. Additionally, analysis of our SHAPE data and previous enzymatic footpinting allows us to propose a model for stem-loop IV function in which tTERT is activated by binding stem IV in the major grove of the helix-capping loop.

Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.

2016-12-23T18:52:10

No abstract

A number of clinical studies have demonstrated correlation between elevated levels of factor IX and the risk of coronary heart disease. Studies by different groups have shown that blocking procoagulant activity of factor IXa could reduce the risk of thrombosis. We propose to study the regulation of factor IXa by phosphatidylserine (PS) which further could open up possibilities to design drugs that might inhibit the up regulation of the procoagulant protein factor IXa. The activated form of factor IX, IXa, forms the Xase complex together with factor VIIIa and Ca2+ on an activated platelet membrane surface. We hypothesize that PS regulates factor IXa by binding to specific sites on factor IXa and thereby enhancing its catalytic activity. We used a soluble form of phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS) as a tool in order to address the role of PS in regulating factor IXa. Intrinsic fluorescence measurements demonstrate that C6PS binds tightly (Kd ~1.3 μM) to and induces changes in conformation of factor IXa. We also monitored the amidolytic activity of 300 nM factor IXa in the presence of C6PS using synthetic substrate Leu-PHG-Arg-pNA. There is a reduction in the amidolytic activity of factor IXa with increasing addition of C6PS and the hyperbolic fit gives an apparent Kd of 130 μM. Recent results show that calcium is required for both amidolytic and proteolytic activity of factor IXa in the presence of C6PS. Our data shows that amidolytic activity of factor IXa in the presence of 600 mM C6PS is saturated at a concentration of calcium near 3 mM. We have also demonstrated that both C6PS and calcium enhance proteolytic activation of factor X by factor IXa. The data shows that as the concentration of Ca2+ increases, the proteolytic activation of factor X by factor IXa increases as well. Enhancement of factor X activation appears to saturate at a calcium concentration of 3 mM. Based on these results, we conclude, 1) C6PS induces a conformational change in factor IXa, 2) C6PS regulates the amidolytic and proteolytic activity of factor IXa, 3) Ca2+ is needed for PS mediated regulation of factor IXa.