Research Interests

 

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Research Interests of the Maurer Laboratory Group

 

Our research is focused on enzymes involved in blood coagulation and related processes.  Members of this critical protein cascade have far reaching effects on wound healing, heart disease, stroke, and cancer.   Further knowledge is still needed on the activation, regulation, and substrate/ligand binding properties of individual enzymes.  In our laboratory, emphasis has been placed on examining the transglutaminase Factor XIII (FXIII), the serine protease thrombin, and the structural protein fibrinogen.  Thrombin is involved in converting fibrinogen into fibrin and in activating a variety of enzymes including FXIII and the protease activated receptors (PARs).  Activated FXIII catalyzes the formation of covalent cross-links within the fibrin blood clot network and other components of the cell. Greater understanding of the biochemical and biophysical features of FXIII, thrombin, and fibrinogen may lead to novel medical strategies to control the actions of these vital proteins and the resultant blood clot architecture

Biochemical basis for cardioprotective Factor XIII V34L and potential roles for Factor XIII V34X Mutants.  Our kinetic and NMR work with FXIII Activation Peptides (28-41) helped to explain why the medically interesting FXIII V34L is more easily activated than the V34 enzyme.  The L34 peptide binds more effectively to the thrombin surface and the conversion to product is more efficient. Studies with other V34X mutants have revealed sequences that can be cleaved more or less readily by thrombin.  Structural NMR studies have then shown that FXIII activation peptides with L34 or F34 exhibit critical interactions with thrombin P36 thus promoting more extensive binding.  Research with anticoagulant thrombins revealed that FXIII AP F34 and W34 can accommodate the loss of thrombin W215 and related residues and still allow for binding and hydrolysis. Studies with intact FXIII V34X proteins are largely in agreement with FXIII V34X (28-41) studies. Furthermore, the intact FXIII V34X studies have demonstrated that regulating FXIII activation may be an effective strategy to help control fibrin clot architecture and its neighboring environments. Selected Refs (1, 8, 12, 14, 18, 23-25, 30, 31)

Elucidating Sources of FXIII Substrate Specificity via Kinetic and NMR Studies  We have focused on elucidating the kinetic and structural features associated with productive binding of glutamine-containing substrates to FXIIIa.  Kinetic and 2D NMR studies have revealed that a series of glutamine-containing peptides encounter a distinctive FXIIIa active site region and that these peptides bind in an extended conformation. Activated Factor XIII (FXIIIa) introduces covalent γ-glutamyl-ε-lysyl crosslinks into the fibrin γ and α chains.  Crosslinks within the fibrin αC region help generate a stiffer clot which is more resistant to fibrinolysis.  Fibrinogen αC (233-425) contains a binding site for FXIIIa and three reactive glutamines. Mass spectrometry and 2D HSQC NMR methods have directly characterized these Qs and probed for sources of FXIIIa substrate specificity. Glycine ethyl ester (GEE) serves as a lysine mimic. Mass spec studies revealed a ranking of Q237 >> Q366 ≥ Q328.  2D HSQC studies with 15N-GEE generated a similar order of reactivities. The Fbg αC E396 residue within the putative FXIII binding segment (389-403) serves as a key anchoring site but is not required for transglutaminase activity. Our results suggest that the reactive glutamines play distinct roles in fibrin crosslinking and clot architecture.  Selected Refs (2, 9, 10, 15, 19, 27)

Activation of Factor XIII is Accompanied by Change in Oligomerization State  Oligomeric assemblies are known to regulate protein function. Factor XIIIA (FXIIIA) is the only transglutaminase found as a homodimeric (A2) zymogen, and it can be activated proteolytically by thrombin-mediated cleavage of the activation peptides (AP) or non-proteolytically by Ca2+ ions. Size exclusion chromatography and analytical ultracentrifugation have been used to study the global rearrangements in FXIIIA2 upon activation. A quantitative assessment revealed that FXIIIA interactions were tight in the dimeric zymogen form. Both non-proteolytic and thrombin-mediated FXIIIA activation resulted in monomeric species. For the first time, direct solution evidence was obtained for the monomeric state of activated FXIIIA in solution. Cleavage of a single AP on FXIII A2 resulted in dissociation and in expression of full enzymatic activity. By contrast, non-proteolytic activation resulted in a less active FXIIIA.  Dimerization of the zymogen is proposed to stabilize FXIIIA in a physiological setting and prevent premature protein cross-linking. Selected Refs (1,7)

Conformational Dynamics of Factor XIII by Hydrogen/Deuterium Exchange and Chemical Modification   We were the first group to utilize amide proton hydrogen to deuterium exchange coupled with MALDI-TOF mass spectrometry (HDX-MS) to probe FXIII solvent exposure upon activation and in the presence of specific ligands.  Segments of the FXIII β-sandwich, catalytic core domain, dimer interface, and β-barrels 1 & 2 undergo changes in solvent exposure upon FXIII activation.  Local and long range effects have been proposed to occur in preparation for substrate binding. This research was later supported by chemical modification studies. The additional conformational influences of the active-site directed ligands iodoacetamide and K9 DON peptide were also examined. Most recently, we probed the conformational influences of different cations. FXIII is known to be susceptible to nonproteolytic activation in a Na+/Ca2+ environment. Overall, these HDX-MS results provided snap shots of FXIII regions that become more or less exposed to solvent in the absence versus presence of ligands. Selected Refs (13, 17, 22, 29)

Identifying and Characterizing Local and Long-Range Effects of Ligand Binding to Thrombin Exosites  The serine protease thrombin contains two anion binding exosites, ABE I and ABE II, that can serve as secondary substrate anchoring sites. Binding to the exosites can also exert local and/or long range effects across the enzyme surface. Thrombin utilizes fibrin binding to ABE I to enhance ability to proteolytically activate FXIII.  NMR methods and HDX-MS have been employed to characterize the binding of ABE II and I directed peptides to thrombin. Peptide ligands based on the fibrinogen γ' chain and the platelet receptor GpIbα bound to ABE II in an extended conformation and exerted both local and long-range effects across thrombin.  Similar HDX effects were observed for ABE I ligands based on the thrombin receptor peptide ligands PAR3 and PAR1.  Most recently, 2D NMR studies have been used to assess the ability of PAR3 and PAR1 ligands to bind to the premature pro-ABE I of prothrombin versus the mature ABE I thrombin. Thrombin is a highly dynamic protein and its conformational environment can respond uniquely to the binding of exosite ligands. Selected Refs (3, 6, 11, 16, 20)

 

Maurer Research Publications:  University of Louisville

  1. Anokhin, B.A., Dean, W.L., Smith, K.A., Flick, M.J., Ariens, RAS, Philippou, H., Maurer, M.C. (2019) “Proteolytic and Nonproteolytic Activation Mechanisms Result in Conformationally and Functionally Different Forms of Coagulation Factor XIII“ FEBS Journal, in press.
  2. Mouapi, KN., Wagner, L.J., Stephens, C.A., Hindi, M.M., Wilkey, D.W., Merchant, M.L., Maurer, M.C. (2019) “Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366)” Thrombosis and Haemostasis 119, 1048-1057.
  3. Billur, R., Sabo, T.M., Maurer, M.C. (2019) “Thrombin Exosite Maturation and Ligand Binding at ABE II Help to Stabilize PAR-Binding Competent Conformation at ABE I” Biochemistry 58, 1048-1060.
  4. Li, B. Billur, R., Maurer, M.C., Kohler, H.P., Raddatz Mueller, P. Alberio, L., Schroeder, V. (2018) “Proline 36 of the Factor XIII Activation Peptide Plays a Crucial Role in Substrate Recognition and Zymogen Activation” Thrombosis and Haemostasis 18, 2037-2045
  5. Hethershaw, E.L., Adamson, P.J., Smith, K.A., Goldsberry. W.N., Pease, R.J., Radford, S.E. Grant, P.J., Ariëns, RAS., Maurer, M.C., Philippou, H. (2018) “The role of beta-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit” Journal of Thrombosis and Haemostasis 16, 1391-1401.
  6. Billur, R., Ban, D., Sabo, T.M., Maurer, M.C. (2017) “Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I” Biochemistry, 56, 6434-6354
  7. Anokhin, B.A., Dean, W.D., Stribinskis, V., Maurer, M.C. (2017) “Activation of Factor XIII is Accompanied by a Change in Oligomerization State" FEBS Journal 284, 3849-3861.
  8. Jadhav, M.A., Goldsberry, W.N., Zink, S.E., Lamb, K.N., Simmons, K.E., Riposo, C.M., Anokhin, B.A., Maurer, M.C. (2017) “Screening Cleavage of Factor XIII V34X Peptides by Thrombin Mutants:  A Strategy for Controlling Fibrin Architecture” BBA:  Proteins and Proteomics 1865, 1246-1254.
  9. Mouapi, K.N., Bell, J.D., Smith, K.A., Ariens, R.A., Philippou H., Maurer, M.C. (2016) “Ranking Reactive Glutamines in the Fibrinogen αC Region That are Targeted by Blood Coagulant Factor XIII” Blood, 127, 2241-8.
  10. Doiphode, P.G., Malovichko, M.V., Mouapi, K.N., Maurer, M.C. (2014) “Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a Matrix-Assisted laser Desorption/Ionization Time-of-Flight Mass Spectrometry Assay” Analytical Biochemistry 457, 74-84.
  11. Malovichko, M.V. Sabo, T.M., Maurer, M.C. (2013) “Ligand Binding to Anion-Binding Exosites Regulates Conformational Properties of Thrombin” J. Biol. Chem 288, 8667-78.
  12. Jadhav, M.A., Lucas, R.C., Goldsberry, W.N., Maurer, M.C. (2011) “Design of Factor XIII V34X Activation Peptides to Control Ability to Interact with Thrombin Mutants” BBA Proteins and Proteomics 1814, 1955-1963.
  13. Woofter, R.T., Maurer, M.C. (2011) “Role of Calcium in the Conformational Dynamics of Factor XIII Activation Examined by Hydrogen-Deuterium Exchange Coupled with MALDI-TOF MS” Arch. Biophys. Biochem. 512, 87-95.
  14. Jadhav, M.A., Isetti, G., Trumbo, T.A., Maurer, M.C.  (2010) “Effects of Introducing Fibrinogen Aα Character into the Factor XIII Activation Peptide Segment” Biochemistry 49, 2918-24.
  15. Cleary, D.B, Doiphode, P.G., Maurer, M.C. (2009) “A Non-Reactive Glutamine Residue of α2-Antiplasmin Promotes Interactions with the Factor XIIIa Active Site Region” J. Thromb. Haemost, 7, 1947-1949.
  16. Sabo, T.M., Maurer, M.C. (2009) “Biophysical Investigation of GpIbα Binding to Thrombin Anion Binding Exosite II” Biochemistry 48, 7110-7122.
  17. Sabo, T.M., Brasher, P.B., Maurer, M.C. (2007) “Perturbations in Factor XIII Resulting From Activation and Inhibition Conditions Examined by Solution Based Methods and Detected by MALDI-TOF MS” Biochemistry 46, 10089-10101.
  18. Isetti, G., Maurer, M.C. (2007) “Employing Mutants to Study Thrombin Residues Responsible for Factor XIII Activation Peptide Recognition:  A Kinetic Study” Biochemistry 46, 2444-2452. 
  19. Cleary, D.B., Maurer, M.C. (2006) “Characterizing the Specificity of Activated Factor XIII for Glutamine Containing Substrate Peptides” Biochim. Biophys. Acta 1764, 1207-1217
  20. Sabo, T.M., Farrell, D.H., Maurer, M.C. (2006) “Conformational Analysis of γ′ Peptide (410-427) Interactions With Thrombin Anion Binding Exosite II” Biochemistry 45, 7434-45.
  21. Maurer, M.C., Trumbo, T.A., Isetti, G., Turner Jr., B.T. (2006) “Probing Interactions Between the Coagulants Thrombin, Factor XIII, and Fibrin(ogen)” Archives of Biochemistry and Biophysics 445, 36 - 45.
  22. Turner Jr., B.T., Sabo, T.M., Wilding, D., Maurer, M.C. (2004) “Mapping of Factor XIII Solvent Accessibility as a Function of Activation State Using Chemical Modification Methods” Biochemistry 43, 9755-65.
  23. Isetti, G. Maurer, M.C. (2004) “Thrombin Activity is Unaltered by N-terminal Truncation of Factor XIII Activation Peptides” Biochemistry 43, 4150-4159.
  24. Isetti, G., Maurer, M.C. (2004) “Probing Thrombin's Ability to Accommodate a V34F Substitution Within the Factor XIII Activation Peptide Segment (28-41)”J. Peptide Research 63, 241-252
  25. Trumbo, T.A. Maurer, M.C. (2003) “V34I and V34A Substitutions Within the Factor XIII Activation Peptide Segment (28-41) Affect Interactions with the Thrombin Active Site.” Thrombosis and Haemostasis. 89, 647-53.
  26. Cleary, D.B., Erhinger, W.D., Maurer, M.C. (2003) “Establishing the Inhibitory Effects of Bradykinin on Thrombin” Archives of Biochemistry and. Biophysics. 410, 96-106. 
  27. Marinescu, A., Cleary, D.B, Littlefield, T.R., Maurer, M.C. (2002) “Structural Features Associated With the Binding of Glutamine-Containing Peptides to Factor XIII” Archives of Biochemistry and Biophysics 406, 9-20.
  28. Cleary, D.B., Trumbo, T.A., Maurer, M.C. (2002) “PAR4-like Peptides Bind to Thrombin Through an Optimized Interaction With the Enzyme Active Site Surface” Archives of Biochemistry and Biophysics 403, 179-188.
  29. Turner, B.T., Maurer, M.C. (2002) “Evaluating the Roles of Thrombin and Calcium in Activation of Coagulation Factor XIII Using H/D Exchange and MALDI-TOF MS” Biochemistry 41, 7947 7954.
  30. Trumbo, T.A., Maurer, M.C. (2002) “Thrombin Hydrolysis of V29F and V34L Mutants of Factor XIII  (28-41) Reveals Roles of the P9 and P4 Positions in Factor XIII Activation” Biochemistry 41, 2859-68.
  31. Trumbo, T.A., Maurer, M.C. (2000) “Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation” J. Biol. Chem. 275, 20627-20631.