Date of Graduation

8-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Sakon, Joshua

Committee Member

Heyes, Colin D.

Second Committee Member

Thallapuranam, Suresh

Third Committee Member

Stites, Wesley E.

Abstract

Clostridium histolyticum secretes collagenases ColG and ColH to cause extensive tissue destruction during myonecrosis. The collagenases are multi-domain enzymes consisting of a N-terminal collagenase module, s1, polycystic kidney disease (PKD)-like domains (s2 in ColG; s2a and s2b in ColH) and collagen-binding domains (CBD) (s3a and s3b in ColG; s3 in ColH). The individual CBD and PKD-like domains chelate calcium to modulate stability and domain rearrangement. Though used by bacteria to break down collagen in the extracellular matrix during infection, therapeutic use of the enzyme to break down excess connective tissue has been approved by the FDA. Meanwhile, the use of the targeting segment to anchor therapeutics at the lesion site is currently underway. To better understand both the mechanism of collagenase during infection and facilitate its use in drug delivery, the high-resolution structures of the PKD-like domains and CBD of collagenases ColG and ColH were solved using X-ray crystallography. The structures of Ca2+-absent (apo)-s2 and s2a, as well as Ca2+-bound (holo)-s2a, s2b, s3a-s3b, and s3 are new structures, while the structure of holo-s3b was re-refined at higher resolution. Individually, the structures of the CBD are similar and share conserved Ca2+ and collagen-binding pockets. In the tandem CBD, s3a-s3b, the domains are related by a pseudo two-fold symmetry that may allow tandem CBD to bind to separate collagen molecules. Furthermore, three aromatic residues that were identified to be crucial to collagen-binding are fully conserved in s3, but only partially conserved in s3a. Evolutionary pressure likely decreased collagen affinity of s3a to prevent s3a-s3b from binding too tightly on the surface of collagen fibril. Meanwhile, the structures of the PKD-like domains are similar, though surface exposed aromatic residues are found only on the ColH PKD-like domains. Furthermore, the change in Cα B-factors upon Ca2+-binding further suggests a divergence of function for ColH and ColG PKD-like domains. In each case, Ca2+-binding further stabilized the robust domains against denaturants heat, urea, and guanidium chloride. For s3a-s3b, the influence of Ca2+ was further explored using small angle X-ray scattering, which indicates the linker between domains gradually contracts in the presence of increasing Ca2+ concentrations.

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