3VV2

Crystal structure of complex form between S324A-subtilisin and mutant Tkpro

Summary for 3VV2

• Entry DOI: 10.2210/pdb3vv2/pdb
• Related: 2z30
• Descriptor: Tk-subtilisin, PROPEPTIDE from Tk-subtilisin, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: hydrolase, proteolysis
• Biological source: Thermococcus kodakarensis; More
• Cellular location: Secreted: P58502 P58502
• Total number of polymer chains: 2
• Total formula weight: 41775.76

Authors

Uehara, R.,Ueda, Y.,You, D.J.,Takano, K.,Koga, Y.,Kanaya, S. (deposition date: 2012-07-12, release date: 2013-03-06, Last modification date: 2024-11-13)

Primary citation

Uehara, R.,Ueda, Y.,You, D.J.,Koga, Y.,Kanaya, S.
Accelerated maturation of Tk-subtilisin by a Leu Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin
Febs J., 280:994-1006, 2013

PubMed Abstract: Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin [Tk-subtilisin in a pro form (Gly1-Gly398)], by autoprocessing and degradation of propeptide [Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)]. The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin. To examine whether the mutation of Leu69 to Pro, which is the most unfavourable residue in the P1 position for subtilisins, affects the maturation of Pro-Tk-subtilisin, the Pro-Tk-subtilisin and Tk-propeptide derivatives with this mutation (Pro-L69P and L69P-propeptide) were constructed and characterized. Pro-L69P was autoprocessed more slowly than Pro-Tk-subtilisin. Nevertheless, it matured to Tk-subtilisin more rapidly than Pro-Tk-subtilisin because L69P-propeptide was degraded by Tk-subtilisin more rapidly than Tk-propeptide. The chaperone function and stability of L69P-propeptide were comparable to those of Tk-propeptide, whereas the inhibitory potency and binding ability of L69P-propeptide were considerably reduced compared to those of Tk-propeptide. The crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) revealed that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation. These results suggest that the Leu→Pro mutation accelerates the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin.

PubMed: 23237738
DOI: 10.1111/febs.12091

Experimental method

X-RAY DIFFRACTION (1.83 Å)