Using Site‐directed Mutagenesis alongside a Collagen binding assay to reveal the role of Polycystic Kidney Disease domain in ColH Collagenase

Hathewaya histolyticum , one of the causative agents of Gas Gangrene and previously known as Clostridium histolyticum , secretes two collagen dismantling enzymes ColG and ColH as part of its pathology. Both ColG and ColH have segmental structure consisting of a catalytic domain, polycystic kidney disease domains (single in ColG; double in ColH) and collagen binding domains (double in ColG; single in ColH). Their ability to seek out and bind to loosely wound regions of collagen make the non‐catalytic portions of ColG and ColH promising drug delivery tools. Crystal structure of dual CBDs found in ColG suggest that ColG may wedge between two collagen molecules to aid in fibril rearrangement and collagen hydrolysis while SAXS derived envelope for ColH PKD‐CBD:[GPRG(POG) 12 ] 3 suggest that ColH interacts with only collagen molecule. This may suggest that the two work together to synergistically degrade collagen. SAXS envelopes of ColH PKD‐CBD:[GPRG(POG) 12 ] 3 show that the PKD domain makes contact with the collagenous peptide. ColH PKD domains 1 and 2 contain regions that are thought to aid in collagen binding for ColH. Collagen binding assay for ColH PKD‐CBD segment bound to type 1 collagen fibrils in a biphasic manner, with one having a higher affinity (K D = 3.39 × 10 −7 M) and the other having a low affinity (K D = 2.11 × 10 −6 M). The PKD‐CBD segment of ColH binds to collagen much tighter than just the CBD (K D = 1.59 × 10 −5 ). Aromatic residues (Tyr780, His782, Tyr796 and Tyr801) located on the potential collagen binding cleft of the PKD domain in Col H PKD‐CBD were mutated to Ser using site directed mutagenesis. In addition an expected tighter binding ColH PKD‐CBD mutant was developed by mutating two non‐aromatic residues (Val778 and Lys785) on the potential collagen binding cleft to Tyr. Val778 and Lys785 were chosen based on a sequence alignment study. Collagen binding assay of the ColH PKD‐CBD four Ser mutant shows a loss of one of the phases of binding and has a KD consistent with the lower affinity phase of the wild type ColH PKD‐CBD (K D = 1.87 × 10 −6 ). Collagen binding assay of the tighter binding ColH PKD‐CBD shows a biphasic plot with the second phase binding much tighter than the wild type (K D = 5 × 10 −8 ). Fusion proteins containing binding segments from ColG and ColH have been used to anchor bFGF and PTH antagonist at their receptors to stimulate healing of bone fractures, enhance bone allografts for spinal fusion and reverse alopecia. Our study would reveal knowledge on how ColH binds to collagen which would help the development of fusion proteins containing ColH binding segments with a higher efficacy. Support or Funding Information National Institutes of Health. Grant Number: 8P30GM103450 Arkansas Biosciences Institute This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .