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. 2018 Sep;285(17):3254-3269.
doi: 10.1111/febs.14611. Epub 2018 Aug 20.

Ca 2+ -Induced Orientation of Tandem Collagen Binding Domains From Clostridial Collagenase ColG Permits Two Opposing Functions of Collagen Fibril Formation and Retardation

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Ca 2+ -Induced Orientation of Tandem Collagen Binding Domains From Clostridial Collagenase ColG Permits Two Opposing Functions of Collagen Fibril Formation and Retardation

Perry Caviness et al. FEBS J. .
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Abstract

To penetrate host tissues, histotoxic clostridia secrete virulence factors including enzymes to hydrolyze extracellular matrix. Clostridium histolyticum, recently renamed as Hathewaya histolytica, produces two classes of collagenase (ColG and ColH). The high-speed AFM study showed that ColG collagenase moves unidirectionally to plane collagen fibril and rebundles fibril when stalled . The structural explanation of the roles for the tandem collagen-binding segment (CBDs) is illuminated by its calcium-bound crystal structure at 1.9 Å resolution (Rwork = 15.0%; Rfree = 19.6%). Activation may involve calcium-dependent domain rearrangement supported by both small-angle X-ray scattering and size exclusion chromatography. At pCa ≥ 5 (pCa = -log[Ca2+ ]), the tandem CBD adopts an extended conformation that may facilitate secretion from the bacterium. At pCa ≤ 4, the compact structure seen in the crystal structure is adopted. This arrangement positions the two binding surfaces ~ 55 Å apart, and possibly ushers ColG along tropocollagen molecules that allow for unidirectional movement. A sequential binding mode where tighter binding CBD2 binds first could aid in processivity as well. Switch from processive collagenolysis to fibril rearrangement could be concentration dependent. Collagen fibril formation is retarded at 1 : 1 molar ratio of tandem CBD to collagen. Tandem CBD may help isolate a tropocollagen molecule from a fibril at this ratio. At 0.1 : 1 to 0.5 : 1 molar ratios fibril self-assembly was accelerated. Gain of function as a result of gene duplication of CBD for the M9B enzymes is speculated. The binding and activation modes described here will aid in drug delivery design.

Accession codes: The full atomic coordinates of the tandem CBD and its corresponding structure factor amplitudes have been deposited in the Protein Data Bank (PDB accession code 5IKU). Small-angle X-ray scattering data and corresponding ab initio models have been submitted to the Small Angle Scattering Biological Data Bank (SASBDB). Accession codes CL2, collagenase module 2, CN2, CP2 are assigned to envelopes for tandem CBD at -log[Ca2+ ] (pCa) 3, 4, 5, and 6, respectively. Accession code DC64 was assigned to the complex of polycystic kidney disease-CBD1-CBD2 with mini-collagen.

Keywords: bacterial collagenase; crystal structure; gene duplication; processive protease; site-directed drug delivery system.

Conflict of interest statement

Competing financial interests

O.M. and J.S. have stock options for BiologicsMD, Inc. K.T. is an emploee of Nippi Inc. R.B., K.J., K.T., J.R.R., O.M., J.S. are co-inventors of USA Provisional Patent 62/457,410.

Figures

Fig. 1
Fig. 1
(a) Phylogenetic tree and domain organization of clostridial collagenases. Amino acid sequences of catalytic modules (CM) from various clostridial collagenases were obtained from DDBJ/EMBL/NCBI databases, and analyzed by neighbor-joining method. Abbreviated names are given for some enzymes. The unit of scale bar is the number of amino acid changes per position. Bootstrap probabilities larger than 70 are shown. The modular structure of respective enzyme is illustrated at right. (b) Sequence alignment of tandem CBD in M9B collagenases from Clostridium sporogenes, C. botulinum A3, C. perfringens, Bacillus. brevis, Paenibacillus dendritiformis, C. sordellii and H. histolytica. CBD1 molecules are aligned on the top rows, and CBD2 molecules are aligned on the bottom rows. Calcium-chelating residues (residues responsible for chelation are in red; residues responsible for positioning the chelators are in orange) and structurally important residues (green) are conserved. The collagen-binding cleft of CBD2 has been carefully mapped [4, 7, 8]. Residues that are expected to interact with collagen are highlighted in blue. One of the most critical residues for collagen interaction in CBD2, Tyr1106, is only conserved in CBD2. Beta-strand A’ is only present in CBD2.
Fig. 2
Fig. 2
Three-dimensional structure alignment of CBD1 (green), CBD2 from the tandem CBD structure (orange), individually solved holo-CBD2 (brown; PDB ID 4HPK), individually solved apo-CBD2 (yellow; PDB ID 1NQJ), and holo-ColH-CBD (salmon; 3JQW). Ca2+ are show as red spheres.
Fig. 3.
Fig. 3.
Crystal structure of tandem CBD and Ca2+ induced domain rearrangement. (a) The CBD1 and the CBD2 are drawn in green and orange, respectively. CBD1 and CBD2 are related by pseudo two-fold rotational symmetry. Four Ca2+ (red) chelates to the CBDs. Aromatic residues (blue) interact with mini-collagen [4, 7, 8]. (b) Size exclusion chromatography and SAXS experiments were carried out in HBS-based calcium buffers of varying pCa. Given the similarity between Ca2+ concentration in the crystal structure and at pCa 3 and 4, four Ca2+ (red) are shown at this pCa. At higher pCa, the exact location of Ca2+ is ambiguous.
Fig. 4
Fig. 4
(a) Retardation of collagen fibril formation by CBD1, n=3. (b) Retardation of collagen fibril formation by CBD2, n=3. (c) Promotion and retardation of collagen fibril formation by tandem CBD, n=3. Error bars represent standard deviations.
Fig. 5
Fig. 5
(a) Surface depiction of the collagen-binding cleft of CBD1. (b) Surface depiction of the collagen-binding cleft of CBD2. Residues that are expected to interact with collagen are highlighted in blue.
Fig. 6.
Fig. 6.
Putative binding mechanism for ColG. (a) SAXS envelop for PKD-CBD1-CBD2 is shown in grey. SAXS envelope for 1:1 complex between PKD-CBD1-CBD2 and mini-collagen is shown in pink. Mini-collagen is shown in blue, PKD is shown in yellow, CBD1 is shown in green, and CBD2 is shown in orange. Ca2+ ions are shown in red. (b) SAXS envelop for CBD1-CBD2 is shown in grey. SAXS envelope for 1:1 complex between CBD1-CBD2 and mini-collagen is shown in pink. Mini-collagen is shown in blue, CBD1 is shown in green, and CBD2 is shown in orange. Ca2+ ions are shown in red.
Fig. 7.
Fig. 7.
Schematic representations of processive collagenolysis by ColG. ColG may intercalate between tropocollagen molecules. CBD2 may bind to either hydrolyzed tropocollagen or non-hydrolyzed tropocollagen. Hydrolysis of collagen moves the molecule towards N-terminus of tropocollagen. CBD2 segment may be the last molecule to be released during the translocation. See text for detail.
Fig. 8
Fig. 8
(a) Experimental scattering profile for CBD1-CBD2 at pCa 3. (b) Experimental scattering profile for CBD1-CBD2 at pCa 4. (c) Experimental scattering profile for CBD1-CBD2 at pCa 5. (d) SAXS scattering profile for CBD1-CBD2 at zero concentration. (e) The Guinier plot for CBD1-CBD2. (f) The interatomic distance distribution function, P(r) for CBD1-CBD2. (g) The Kratky plot for CBD1-CBD2. (h) The Porod-Debye plot for CBD1-CBD2.
Fig. 9
Fig. 9
(a) Experimental scattering profile for PKD-CBD1-CBD2. (b) SAXS scattering profile for PKD-CBD1-CBD2 at zero concentration. (c) The Guinier plot for PKD-CBD1-CBD2. (d) Experimental scattering profile for PKD-CBD1-CBD2-[(POG)10]3. (e) SAXS scattering profile for PKD-CBD1-CBD2-[(POG)10]3 at zero concentration. (f) The Guinier plot for PKD-CBD1-CBD2-[(POG)10]3. (g) The interatomic distance distribution function, P(r) for PKD-CBD1-CBD2 (dotted line) vs. PKD-CBD1-CBD2-[(POG)10]3 (solid line). (h) The Kratky plot for PKD-CBD1-CBD2 (dotted line) vs. PKD-CBD1-CBD2-[(POG)10]3 (solid line). (i) The Porod-Debye plot for PKD-CBD1-CBD2 (dotted line) vs. PKD-CBD1-CBD2-[(POG)10]3 (solid line).
Fig. 10
Fig. 10
(a) SAXS scattering profile for CBD1-CBD2-[(POG)10]3 at zero concentration. (b) The Guinier plot for CBD1-CBD2-[(POG)10]3. (c) The interatomic distance distribution function, P(r) for CBD1-CBD2 (dotted line) vs. CBD1-CBD2-[(POG)10]3. (d) The Kratky plot for CBD1-CBD2-[(POG)10]3 (solid line). (e) The Porod-Debye plot for CBD1-CBD2-[(POG)10]3.

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