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. 2024 Apr 16;121(16):e2321002121.
doi: 10.1073/pnas.2321002121. Epub 2024 Apr 9.

A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen

Jamil Serwanja  1   2 , Alexander C Wieland  1   2 , Astrid Haubenhofer  1   2 , Hans Brandstetter  1   2 , Esther Schönauer  1   2
Affiliations

A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen

Jamil Serwanja et al. Proc Natl Acad Sci U S A. .

Abstract

Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium, Bacillus, Spirochaetes, and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

Keywords: Clostridium; bacterial enzyme; collagen degradation; collagenase; virulence factor.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Binding of ColG variants to fibrillar collagen, soluble collagen, and gelatin. (A) Schematic domain organization of ColG and ColH from H. histolytica, Ghcol from Grimontia hollisae, and VhaC from Vibrio harveyi VHJR7 (23, 27, 28). AD (dark blue), linker (light green), PD (light blue), PKD (yellow), CBD (orange), and bacterial prepeptidase C-terminal domain (orange striped) (PPC). (B) Ribbon representation of the CU of ColG. The catalytic zinc ion (gray) and the catalytic residues (light orange) are shown in ball-and-stick representation. The linker is highlighted (green). Molecular figures were created with PyMOL (29). (C) Release of fluorescently labeled ColG variants from fibrillar collagen. (D) Binding affinities to soluble collagen and gelatin. Apparent dissociation constants were determined via indirect ELISA, whereby the apparent Kd values represent the binding to gelatin or soluble collagen each with multiple binding sites per molecule. Due to very weak binding affinities, no Kd values could be determined for ColG-PKD. The binding curves are shown in SI Appendix, Fig. S1 and Kd values are given in SI Appendix, Table S1. nd, could not be determined.
Fig. 2.
Fig. 2.
ColG does not unwind soluble collagen globally. (A) Thermal transition curves and melting temperatures from CD experiments for soluble collagen with or without ColG-CU G494V and α-chymotrypsin. (B) SDS-PAGE analysis of a degradation complementation assay, in which soluble collagen was incubated with equimolar concentrations of ColQ1-PD and increasing concentrations of ColQ1-CU G472V at 25 °C. The integrity of the substrate was verified by coincubation with 0.83 μM α-chymotrypsin. (C–F) V-(GXY)26 is a short collagen-like substrate. (C) SDS-PAGE analysis of time course of incubation of V-(GXY)26 with and without ColG-CU WT, ColG-CU G494V, and α-chymotrypsin. Note that the V-domain migrates as an SDS-stable trimer at an apparent mass of 30 kDa; similarly, V-(GXY)26 migrates as a trimer. (D) CD spectrum of V-(GXY)26 and of (GXY)26, prepared by pepsin digest of VScl2.3-(GXY)26. (E and F) Melting profiles of V-(GXY)26 (E) and of the single V-domain (F) determined by CD spectroscopy. (G) CD spectra of V-(GXY)26 recorded at 25 and 35 °C. The inlet shows the CD difference spectrum of the 25 °C spectrum minus the 35 °C spectrum.
Fig. 3.
Fig. 3.
Local and transient triple-helicase activity of AD requires simultaneous colocalization of the PD for collagen cleavage. (A) Melting profiles of the short collagen mimic V-(GXY)26 in absence or presence of ColG-CU G494V, ColG-AD, and ColG-CBD2 monitored via CD spectroscopy. (B) Melting temperatures of V-(GXY)26 in absence or presence of ColG-CU G494V, ColG-AD, and ColG-CBD2 (mean ± SD). (C) SDS-PAGE analysis of time course of coincubation of 3.33 µM soluble collagen with increasing concentrations of ColQ1-AD (1 to 3 µM) in the presence of 0.6 µM ColQ1-PD at 25 °C. Minor fragments are highlighted with an asterisk. (D and E) SDS-PAGE analysis of time course of coincubation of 10 µM V2.28-(GXY)26, which migrates as monomer, with 1, 5 or 10 µM ColG-AD-MBP (D) or 1, 5 or 10 µM ColG-CU G494V (E) in the presence of 10 µM ColG-PD. Control reactions can be found in SI Appendix, Fig. S7.
Fig. 4.
Fig. 4.
Effect of single-point mutations in AD on soluble collagen degradation, binding affinities toward gelatin and soluble collagen, and on triple-helicase activity. (A) Activity of ColQ1-CU WT and its mutants toward soluble collagen. SDS-PAGE analysis of time course of 3.33 µM soluble collagen coincubation with 0.2 µM ColQ1-CU variants at 25 °C. (B and C) Kd values of ColQ1-CU G472V (=WT) and mutants were determined by indirect ELISA. Binding affinities toward gelatin (B) were tested at 37 °C, while binding affinities toward soluble collagen (C) were tested at 25 °C to preserve the triple-helical fold. Loss of affinity was marked with a magenta bar if no binding at 100 µM concentration was detected. Mutant N226A showed no saturable binding to both substrates up to 118 µM. Therefore, its Kd values were estimated to be >150 µM (striped). (D) Change in melting temperature of V-(GXY)26 in the presence of ColG-CU G494V (=WT) or its mutants determined by CD spectroscopy. Homologous variants of ColQ1 and ColG are arranged on top of each other (B–D) in the figure. (E) Table of the homologous residues in ColQ1-AD and ColG-AD that were investigated. (F) Ribbon and surface representation of the AD. Residues important for soluble collagen binding, i.e., F148, E191, Y198, and N251 (magenta), and the residues crucial for unwinding, i.e., R194, Y201, and F295 (gray), are highlighted as sticks. To ease comparison between the panels, the residues involved in collagen binding (magenta circle) and unwinding (gray triangle) are marked.
Fig. 5.
Fig. 5.
Conformational trapping and its effect on collagen degradation and binding. (A) Scheme of crosslinked mutants. (B) Model of (semi)-closed conformation of ColG-CU. Mutation sites for the introduction of cysteines are shown in sticks. (C–E) SDS-PAGE analysis of time course of degradation of soluble collagen by ColG-CU variants in absence (C and D) or presence (E) of 10 mM ß-mercaptoethanol. (F) Binding affinity of ColG-CU G494V (=WT G494V) and the mutants were determined by microscale thermophoresis. The binding curves can be found in SI Appendix, Fig. S15. (G) Change in melting temperature of the collagen mimic V-(GXY)26 in the presence of WT G494V and the mutants determined by CD spectroscopy. Experiments (F and G) were performed in the presence or absence of 1 mM ß-mercaptoethanol. (H) Binding to fibrillar collagen monitored via ligand release assay using fluorescently labeled, inactive ColG-CU. (I) Degradation of fibrillar collagen by crosslinked variants monitored via fluorescamine-citrate assay. Experiments (H and I) were performed in the presence or absence of 10 mM ß-mercaptoethanol.
Fig. 6.
Fig. 6.
Schematic model of CU binding to soluble and fibrillar collagen illustrating antagonistic and synergistic effects. The PD sterically restricts access to AD binding sites in the saddle-shaped CU for soluble collagen molecules and fibrillar collagen (illustrated by a microfibril). Only suitably preoriented molecules can access and bind, resulting in an overall reduction of kon. Compared to the AD, the affinity of the CU toward soluble collagen is lowered, because the presence of the PD hardly diminishes koff for the relatively small triple helix, whereas the saddle-shaped CU can “cage” the considerably larger microfibril, resulting in a substantial koff-effect and thus in an overall higher affinity of the CU for microfibrils.
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