The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing Within a Symmetric Complex



Sneha Singh1, Alexis Nazabal2, Senthilvelrajan Kaniyappan3, Jean-Luc Pellequer4, Alisa S. Wolberg5, Diana Imhof6, Johannes Oldenburg1, Arijit Biswas1


  1. Institute of Experimental Hematology and Transfusion medicine, Sigmund-Freud Street 25, University Hospital of Bonn. 53127, Bonn, Germany
  2. CovalX, Schützengasse 2, CH-8001 Zürich, Switzerland
  3. German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Str. 27, 53127, Bonn, Germany
  4. University of Grenoble Alpes, CEA, CNRS, IBS, F-38000, Grenoble, France
  5. Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 8018A Mary Ellen Jones Building, Chapel Hill, North Carolina 27599-7035, United States
  6. Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany


Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with 2 catalytic A subunits and 2 carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B-subunits, activated FXIII (FXIIIa) covalently cross-links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamic simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly and/or activity to reduce bleeding and thrombotic complications.

CovalX Technology Used

Complex Tracker


The purified FXIII-A2B2 complex was mixed with the CovalX K200 Stabilization kit and then 1 μL of each sample was spotted on a MALDI plate. The plate was crystallized at room temperature and placed into a MALDI mass spectrometer that had been modified with the CovalX HM2 high mass detection system. Following the sample analysis, the data were analyzed using the CovalX Complex Tracker software.

The XL-MS was used to identify both inter and intra-molecular connections that are found within the FXIII-A2B2 complex. The FXIII subunits and complex were analyzed separately in order to define the residues found in the FXIII-A2B2 inter-subunit interface. From this analysis, 358 total peptides were found and these contributed to 80% coverage for FXIII-A and 91% coverage for FXIII-B. Within the FXIII-A2B2 heterotetramer, there were 34 cross-linked peptides found in the residues from the FXIII-A C-terminal barrel domains and the FXIII-B N-terminal S1, S2, and S3 sushi domains. The residues in the FXIII-A N-terminal β-sandwich domain were discovered to be cross-linked with the residues from the FXIII-B C-terminal S6, S7, S8, and S9 sushi domains. FXIII-B sushi domains S3, S4, S5, S7, S8, and S9 were cross-linked to the catalytic core region of the FXIII-A. Intra-subunit crosslinks were also found with the FXIII-B2 dimer interface and residues in the N-terminal sushi domains (S1-S4) with fewer found in S6, S7, and S8. The XL-MS data helped to contribute to the conclusion made by researchers that there is a new view of the FXIII complex as well as new mechanisms that explain association and disassembly.


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