Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry

Song, Hongjian; Olsen, Ole H.; Persson, Egon; Rand, Kasper D.

doi:10.1074/jbc.m114.614297

Cited by 20 publications

(29 citation statements)

References 36 publications

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“…At the completion of this study, we conclude that whole‐protein alanine‐scanning mutagenesis is a useful tool to identify protein residues that potentially contribute to allosteric mechanisms. In particular, this probing approach is likely to be complementary to the protein backbone monitoring offered by hydrogen/deuterium exchange (Beckett, ; Frantom, Zhang, Emmett, Marshall, & Blanchard, ; Landgraf et al., ; Prasannan et al., ; Song, Olsen, Persson, & Rand, ; Underbakke et al., ). This independent method will also be beneficial to guide structural comparisons to focus on which regions of a protein might experience allosterically relevant conformational and/or dynamic changes.…”

Section: Discussionmentioning

confidence: 99%

Whole‐protein alanine‐scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism

Tang

Fenton

2017

Human Mutation

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Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play “key” roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid side-chains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (activator) and alanine (inhibitor). Each non-alanine/non-glycine residue hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using limited numbers of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms.

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Section: Discussionmentioning

confidence: 99%

Whole‐protein alanine‐scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism

Tang

Fenton

2017

Human Mutation

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“…This effect became even more pronounced in the presence of sTF with the cofactor still able to mature the S1 pocket to FVIIa-WT levels through the proposed pathway II (10) but unable to potentiate amidolytic activity. The unfavorable conformation of the TF-binding helix may explain this as it is likely to result in the low extent of N terminus insertion, which without the effects of Tyr 172 results in a signif- icant destabilization of the whole activation pocket and the active site (5 (20) where an increase in Trp 215 backbone amide protection was seen upon sTF addition. The combined approach of SASA calculation and in-solution quenching used here allowed an elaboration on these observations.…”

Section: Discussionmentioning

confidence: 99%

“…The crystal structure of FVIIa-Y T in complex with sTF revealed that Tyr 172 , as in trypsin (45) (20). In addition, the hydroxyl group of Tyr 172 displaces a water molecule (HOH 1 ; Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies with thrombin, another trypsin-like serine protease, reveal a highly plastic protease fold for the apo form of thrombin that undergoes structural transitions upon cofactor binding (13)(14)(15)(16)(17)(18)(19). In particular, segment 215-217 was shown to be vital for substrate access and cofactor-mediated allosteric regulation, which may also be the case in TF-mediated allosteric regulation of FVIIa activity (20,21).…”

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confidence: 99%

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Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

Sørensen

Madsen

Svensson

et al. 2016

Journal of Biological Chemistry

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The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

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“…49,50 Reduced N-terminal hydrogen-deuterium exchange upon TF binding to FVIIa supports the hypothesis that the N-terminal Ile 153 is not fully inserted into the activation pocket when free in solution. 51 Additionally, unlike most other serine proteases, oxyanion hole formation in free FVIIa does not occur upon proteolytic activation, but instead upon substrate interaction. 52 As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, 52 allowing it to circulate with a half-life of 90 minutes.…”

Section: Structure Tissue Factormentioning

confidence: 99%

Structure–Function Relationship of the Interaction between Tissue Factor and Factor VIIa

Gajsiewicz

Morrissey

2015

Semin Thromb Hemost

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Interactions between tissue factor and factor VIIa are the primary initiators of coagulation in hemostasis and certain thrombotic diseases. Tissue factor, an integral membrane protein expressed extensively outside of the vasculature, is the regulatory protein cofactor for coagulation factor VIIa. Factor VIIa, a trypsin-like serine protease homologous with other blood coagulation proteases, is weakly active when free in solution and must bind its membrane-bound cofactor for physiologically-relevant activity. Tissue factor allosterically activates factor VIIa by several mechanisms such as active site positioning, spatial stabilization, and direct interactions with the substrate. Protein-membrane interactions between tissue factor, factor VIIa, and substrates all play critical roles in modulating the activity of this enzyme complex. Additionally, divalent cations such as Ca2+ and Mg2+ are critical for correct protein folding, as well as protein-membrane and protein-protein interactions. The contributions of these factors towards tissue factor-factor VIIa activity are discussed in this review.

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Sites Involved in Intra- and Interdomain Allostery Associated with the Activation of Factor VIIa Pinpointed by Hydrogen-Deuterium Exchange and Electron Transfer Dissociation Mass Spectrometry

Cited by 20 publications

References 36 publications

Whole‐protein alanine‐scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism

Whole‐protein alanine‐scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

Structure–Function Relationship of the Interaction between Tissue Factor and Factor VIIa

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