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Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Structures of factor XI and prekallikrein bound to domain 6 of high molecular weight kininogen reveals alternate domain 6 conformations and exosites Chan Li , Awital Bar Barroeta , Szu Shen Wong , Hyo Jung Kim , Monika Pathak , Ingrid Dreveny , Joost C. M. Meijers , Jonas Emsley Journal Of Thrombosis And Haemostasis DOI: 10.1016/j.jtha.2023.03.042 Diamond Proposal Number(s): [26803] Open Access Show Abstract ▼ Abstract: Background: High molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both substrate and co-factor for activated plasma kallikrein (PKa) and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK. Objective: To determine the structure of the complex formed between PK apple domains and a HKD6 fragment and compare this to the FXI-HK complex. Methods: We produced recombinant FXI and PK heavy chains (HC) spanning all four apple domains. We co-crystallised PKHC (and subsequently FXIHC) with a 31 amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analysed the full length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry (HDX-MS). Results and conclusions: The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3. The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.	Apr 2023
I04-Macromolecular Crystallography	Chasing the structural diversity of the transcription regulator Mycobacterium tuberculosis HigA2 William Richardson , Gyun Won Kang , Hee Joong Lee , Kang Mu Kwon , Saron Kim , Hyo Jung Kim Iucrj 8 DOI: 10.1107/S2052252521007715 Diamond Proposal Number(s): [19880] Open Access Show Abstract ▼ Abstract: Transcription factors are the primary regulators of gene expression and recognize specific DNA sequences under diverse physiological conditions. Although they are vital for many important cellular processes, it remains unclear when and how transcription factors and DNA interact. The antitoxin from a toxin–antitoxin system is an example of negative transcriptional autoregulation: during expression of the cognate toxin it is suppressed through binding to a specific DNA sequence. In the present study, the antitoxin HigA2 from Mycobacterium tuberculosis M37Rv was structurally examined. The crystal structure of M. tuberculosis HigA2 comprises three sections: an N-terminal autocleavage region, an α-helix bundle which contains an HTH motif, and a C-terminal β-lid. The N-terminal region is responsible for toxin binding, but was shown to cleave spontaneously in its absence. The HTH motif performs a key role in DNA binding, with the C-terminal β-lid influencing the interaction by mediating the distance between the motifs. However, M. tuberculosis HigA2 exhibits a unique coordination of the HTH motif and no DNA-binding activity is detected. Three crystal structures of M. tuberculosis HigA2 show a flexible alignment of the HTH motif, which implies that the motif undergoes structural rearrangement to interact with DNA. This study reveals the molecular mechanisms of how transcription factors interact with partner proteins or DNA.	Sep 2021
I24-Microfocus Macromolecular Crystallography	Crystal structure and substrate-induced activation of ADAMTS13 Anastasis Petri , Hyo Jung Kim , Yaoxian Xu , Rens De Groot , Chan Li , Aline Vandenbulcke , Karen Vanhoorelbeke , Jonas Emsley , James T. B. Crawley Nature Communications 10 DOI: 10.1038/s41467-019-11474-5 Diamond Proposal Number(s): [19880] Open Access Show Abstract ▼ Abstract: Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.	Aug 2019

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