Tracing an allosteric pathway regulating the activity of the HslV protease

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 111; no. 6; pp. 2140 - 2145
• Main Authors: Shi, Lichi, Kay, Lewis E
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 11.02.2014; National Acad Sciences
• Subjects: Allosteric Regulation; Binding sites; Biological Sciences; Cells; Endopeptidase Clp - chemistry; Endopeptidase Clp - metabolism; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Homeostasis; Models, Molecular; Molecular structure; Nuclear Magnetic Resonance, Biomolecular; Proteases; Proteins; Proteolysis; Substrate Specificity; protein NMR; molecular machines; protein dynamics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1318476111

The HslU–HslV complex functions as a bacterial proteasome, degrading substrate polypeptides to preserve cellular homeostasis. Here, we use methyl-Transverse Relaxation-Optimized Spectroscopy (TROSY) and highly deuterated, methyl-protonated samples to study the 230 kDa dodecameric HslV protease component that is structurally homologous to the stacked pair of β ₇-rings of the proteasome. Chemical shift assignments for over 95% of the methyl groups are reported. From the pH dependence of methyl chemical shifts, a pK ₐ of 7.7 is measured for the amine group of the catalytic residue T1, confirming that it can act as a proton acceptor during the initial step in substrate proteolysis. Analyses involving a series of single site mutants in HslV, localized to HslU binding sites or regions undergoing significant changes on HslU binding, have identified hot spots whose perturbation leads to an allosteric pathway of propagated changes in structure and ultimately, substrate proteolysis efficiency. HslV plasticity is explored through methyl-TROSY ¹³C relaxation dispersion experiments that are sensitive to millisecond timescale dynamics. The data support a dynamic coupling between residues involved in both HslU and substrate binding and residues localized to the active sites of HslV that facilitate the allostery between these distal sites. An important role for dynamics has also been observed in the archaeal proteasome, suggesting a more generally conserved role of motion in the function of these barrel-like protease structures.