Metal ion stabilization of the conformation of a recombinant 19‐kDa catalytic fragment of human fibroblast collagenase

Abstract

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.

Molecular Interactions between Matrilysin and the Matrix Metalloproteinase Inhibitor Doxycycline Investigated by Deuterium Exchange Mass Spectrometry

“…Members of this family typically contain an autoinhibitory pro domain, a catalytic zinc-binding domain that operates via a "cysteine switch" mechanism (Wart and Birkedal-Hansen, 1990), and a substrate-binding hemopexin-like domain (Visse and Nagase, 2003). In addition, MMPs contain a second zinc atom and at least two calcium atoms that are not part of the catalytic center and seem to play roles in stabilizing tertiary structure (Lowry et al, 1992;Willenbrock et al, 1995). MMPs are expressed as zymogens (pro-MMPs), and are either secreted from the This work was supported by National Institutes of Health grants 2-R01-HL43617-10A2 (to F.V.)…”

Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein

“…17; a full list is included in supplemental Table 1). These proteins with highly charged binding sites share common biological functions for conformational switching or calcium titration; for example, calsequestrin (58), human Hsp70 (59), and an Na ϩ /Ca 2ϩ exchanger binding domain (60) have a similarly high charge density binding pocket as Protein S. Many of these proteins with exceptionally electronegative binding sites have already been reported to harbor great structural instability in the absence of calcium (54,55,(57)(58)(59)(60)(61)(62) or have increases in folding speed due to calcium (63). Because many of these proteins are also multidomain, our results on Protein S may apply to many of the proteins with high charge density sites.…”

Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion

“…This fluctuation of calcium concentrations can alter cellular physiology by means of structural changes induced by calciumbinding proteins, such as the calcium-mediated electrostatic switch mechanism for m-calpain [24]. Binding of Ca 2+ ions to proteins may confer structural stability in which reagent-induced and thermal denaturation [25,26] are less probable, or modulate substrate binding [27].…”

“…1 To whom correspondence should be addressed (email sang@chem.fsu.edu). ions stabilize MMP-1 [25], mediate signal transduction in the expression of MMP-2 [36], facilitate the activation of pro-MMP-3 [37], and induce cleavage of both N-and C-termini, resulting in activation and C-terminal truncation of pro-MMP-9 from the complex of pro-MMP-9 and chondroitin sulfate proteoglycans [38], and that Ca 2+ ion influx inhibits MT1-MMP processing [39]. However, little research has focused on the structural and func- …”