How Thrombomodulin Enables W215A/E217A Thrombin to Cleave Protein C but Not Fibrinogen.

The W215A/E217A mutant thrombin is called "anticoagulant thrombin" because its activity toward its procoagulant substrate, fibrinogen, is reduced more than 500-fold whereas in the presence of thrombomodulin (TM) its activity toward its anticoagulant substrate, protein C, is reduced less than 10-fold. To understand how these mutations so dramatically alter one activity over the other, we compared the backbone dynamics of wild type thrombin to those of the W215A/E217A mutant thrombin by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). Our results show that the mutations cause the 170s, 180s, and 220s C-terminal ß-barrel loops near the sites of mutation to exchange more, suggesting that the structure of this region is disrupted. Far from the mutation sites, residues at the N-terminus of the heavy chain, which need to be buried in the Ile pocket for correct structuring of the catalytic triad, also exchange much more than in wild type thrombin. TM binding causes reduced H/D exchange in these regions and also alters the dynamics of the ß-strand that links the TM binding site to the catalytic Asp 102 in both wild type thrombin and in the W215A/E217A mutant thrombin. In contrast, whereas TM binding reduces the dynamics the 170, 180 and 220 s C-terminal ß-barrel loops in WT thrombin, this region remains disordered in the W215A/E217A mutant thrombin. Thus, TM partially restores the catalytic activity of W215A/E217A mutant thrombin by allosterically altering its dynamics in a manner similar to that of wild type thrombin.

Hydrogen/Deuterium Exchange and Nuclear Magnetic Resonance Spectroscopy Reveal Dynamic Allostery on Multiple Time Scales in the Serine Protease Thrombin.

A deeper understanding of how hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals allostery is important because HDX-MS can reveal allostery in systems that are not amenable to nuclear magnetic resonance (NMR) spectroscopy. We were able to study thrombin and its complex with thrombomodulin, an allosteric regulator, by both HDX-MS and NMR. In this Perspective, we compare and contrast the results from both experiments and from molecular dynamics simulations. NMR detects changes in the chemical environment around the protein backbone N-H bond vectors, providing residue-level information about the conformational exchange between distinct states. HDX-MS detects changes in amide proton solvent accessibility and H-bonding. Taking advantage of NMR relaxation dispersion measurements of the time scale of motions, we draw conclusions about the motions reflected in HDX-MS experiments. Both experiments detect allostery, but they reveal different components of the allosteric transition. The insights gained from integrating NMR and HDX-MS into thrombin dynamics enable a clearer interpretation of the evidence for allostery revealed by HDX-MS in larger protein complexes and assemblies that are not amenable to NMR.

Evaluating the Sulfate Radical Anion as a New Reagent for In-Cell Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) has demonstrated the ability to inform on the higher order structure of proteins. Recent technological advances have extended FPOP to live cells (IC-FPOP) using multiple cell lines and in vivo (IV-FPOP) using C. elegans. These innovations allow proteins to be studied in their native cellular environment. Hydroxyl radicals are generated via the photoloysis of hydrogen peroxide. Hydrogen peroxide is a signaling molecule that can induce changes to some proteins in the cell limiting the proteins that can be studied by IC-FPOP. Here, we evaluate the sulfate radical anion as a footprinting label in IC-FPOP with sodium persulfate as the precursor. Our findings show a 1.5-fold increase in the number of modified proteins compared to IC-FPOP using hydroxyl radicals at the same precursor concentration demonstrating the amenability of this radical with IC-FPOP.

Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex.

Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-ß-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced µs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased µs-ms dynamics in a ß-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two ß-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

Structural and Functional Characterization of Dynamic Oligomerization in Burkholderia cenocepacia HMG-CoA Reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.

Accurate Prediction of Amide Exchange in the Fast Limit Reveals Thrombin Allostery.

Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) of proteins has become extremely popular for identifying ligand-binding sites, protein-protein interactions, intrinsic disorder, and allosteric changes upon protein modification. Such phenomena are revealed when amide exchange is measured in the fast limit, that is, within a few minutes of exchange in deuterated buffer. The HDXMS data have a resolution of the length of peptides and are difficult to interpret because many different phenomena lead to changes in hydrogen/deuterium exchange. We present a quantitative analysis of accelerated molecular dynamics simulations that provides impressive agreement with peptide-length HDXMS data. Comparative analysis of thrombin and a single-point mutant reveals that the simulation analysis can distinguish the subtle differences in exchange due to mutation. In addition, the results provide a deeper understanding of the underlying changes in dynamics revealed by the HDXMS that extend far from the site of mutation.

Dynamic Consequences of Mutation of Tryptophan 215 in Thrombin.

Thrombin normally cleaves fibrinogen to promote coagulation; however, binding of thrombomodulin to thrombin switches the specificity of thrombin toward protein C, triggering the anticoagulation pathway. The W215A thrombin mutant was reported to have decreased activity toward fibrinogen without significant loss of activity toward protein C. To understand how mutation of Trp215 may alter thrombin specificity, hydrogen-deuterium exchange experiments (HDXMS), accelerated molecular dynamics (AMD) simulations, and activity assays were carried out to compare the dynamics of Trp215 mutants with those of wild type (WT) thrombin. Variation in NaCl concentration had no detectable effect on the sodium-binding (220sCT) loop, but appeared to affect other surface loops. Trp215 mutants showed significant increases in amide exchange in the 170sCT loop consistent with a loss of H-bonding in this loop identified by the AMD simulations. The W215A thrombin showed increased amide exchange in the 220sCT loop and in the N-terminus of the heavy chain. The AMD simulations showed that a transient conformation of the W215A thrombin has a distorted catalytic triad. HDXMS experiments revealed that mutation of Phe227, which engages in a π-stacking interaction with Trp215, also caused significantly increased amide exchange in the 170sCT loop. Activity assays showed that only the F227V mutant had wild type catalytic activity, whereas all other mutants showed markedly lower activity. Taken together, the results explain the reduced pro-coagulant activity of the W215A mutant and demonstrate the allosteric connection between Trp215, the sodium-binding loop, and the active site.

Kinetic characterization of an oxidative, cooperative HMG-CoA reductase from Burkholderia cenocepacia.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different Vmax values.