Insights into ClpXP proteolysis: heterooligomerization and partial deactivation enhance chaperone affinity and substrate turnover in Listeria monocytogenes † †Electronic supplementary information (ESI) available: Figures, tables and experimental procedures. See DOI: 10.1039/c6sc03438a Click here for

Caseinolytic protease from Listeria exploits two paths of proteolytic stimulation: heterooligomerization and partial inhibitor binding both enhance ClpX chaperone affinity.


Supplementary
Structural superposition of LmClpP2 from LmClpP1/2 heterocomplex (PDB code 4RYF, blue) and SaClpP (PDB code 3V5E, gray). Residues of the catalytic triad are shown in cyan for LmClpP2 and dark gray for SaClpP, heteroatoms O and N are shown in red and blue, respectively. The figure was made with PyMOL 1.3. 1

Supplementary Tables
Table S1 Names and abbreviations of non-natural amino acids used in the peptidase substrate library screening.

Abbreviation
Name D-Ala
LmClpP1 wt /2 wt , LmClpP1 wt /2 m , LmClpP1 m /2 wt and LmClpP1 wt /2 wt heterocomplex variants were overexpressed and purified as detailed previously. 4 In short, C-terminally Strep-II-tagged LmClpP1 and C-terminally His6-tagged LmClpP2 were introduced into pETDuet-1 vector. Tagfree SaClpX was purified as described previously. 5 In short, for the overexpression of SaClpX with an N-terminal His6-tag and TEV site pET301 vector was used in E. coli BL21(DE3) cells. The cell lysate was loaded on a HisTrap HP column (GE Healthcare). TEV protease and 1 mM EDTA were added to the pooled fractions. After cleavage and removal of imidazole, the protein solution was loaded on a HisTrap HP column and the flow-through was collected which was further purified by gel filtration.
N-terminally Strep-II-tagged eGFP with a C-terminal SsrA tag (AGKEKQNLAFAA for L. monocytogenes and AANDENYALAA for E. coli) was overexpressed in E. coli KY2266 (ΔclpXP, Δlon, ΔhslVU) 6 using pDEST007 expression vector and purified by affinity chromatography and gel filtration as described previously. 4,5 GlyA (UniProt entry Q8Y4B2) and PncB (UniProt entry Q8Y826) with an N-terminal Strep-II tag and a Cterminal LmSsrA tag (AGKEKQNLAFAA) were constructed from L. monocytogenes EGD-e genomic DNA S12 in pDEST007 plasmid vector with Gateway® Technology using the primers listed in Table S5. The plasmids were transformed into E. coli SG1146a cells. 2 L LB culture was induced with 0.2 μg/mL anhydroteracycline after reaching an OD600 of 0.6. GlyA was incubated at 37 °C for 5 hours and PncB at 25 °C over night. Cells were harvested by centrifugation, washed with PBS, resuspended in PBS and ultrasonicated on ice. The cell lysate was cleared by centrifugation (38,000 g, 40 min, 4 °C) and loaded on a pre-equlibrated 5 mL StrepTrap HP column (GE Healthcare). The column was washed with 6 CV binding buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) and eluted with 5 CV binding buffer + 2.5 mM desthiobiotin. EcRpoS and SaGudB with SsrA tags (AANDENYALAA and AGKSNNNFAVAA, respectively) were purified similarly as described elsewhere. 7 Creatine kinase (10 127 566 001), lactate dehydrogenase (10 128 155 001) and pyruvate kinase (10 127 876 001) were purchased from Roche.

Peptidase assay
In the substrate library screening following fluorogenic tripeptide substrate libraries were used: Ac-Ala-hArg-Xaa-ACC for P1 site, Ac-Ala-Xaa-Leu-ACC for P2 site and Ac-
The samples were desalted using reverse phase extraction cartridges (tC18 SepPak, 50 mg, Waters) on a vacuum manifold. Cartridges were washed three times with 1 mL acetonitrile (ACN), 1 mL 50% ACN with 0.5% formic acid (FA) and twice with 1 mL 0.1% FA. After addition of FA to a final concentration of 0.5% FA, the samples were loaded on the cartridges, washed with 1 mL 0.1% FA and twice with 1 mL 0.5% FA. Peptides were eluted into low binding reaction tubes (Eppendorf) with 3×200 μL 80% ACN with 0.5% FA. The eluates were vacuum dried. The dried samples were dissolved in 100 μL 1% FA, ultrasonicated for 15 min and filtered on a 0.45 μm pore size filter.
LC-MS/MS analysis was carried out on a Dionex UltiMate 3000 nano HPLC coupled to a Thermo Finnigan Orbitrap XL. Samples were loaded onto a C18 NanoTrap Column (Acclaim C18 PepMap100, 2 cm×10 μm i.d., 5 μm particle size, 300 Å pore size) and separated on a Dionex C18 PepMap RSLC (Acclaim C18 PepMap RSLC, 50 cm×75 μm i.d., 2 μm particle size, 100 Å pore size) column. Solvent A consisted of water + 0.1% FA + 5% DMSO and solvent B consisted of ACN + 0.1% FA + 5% DMSO. Separation was achieved at a constant flow rate of 0.2 µL/min using a gradient from 4% B to 30% B over 90 min and a subsequent wash-out to 80% B over 33 min. Full scans were carried out with an m/z range of 350 -1400 at a resolution of 60000 followed by a TOP5 CID fragmentation step (35 eV collision energy, activation time: 30 ms) using dynamic exclusion (30 s).
Fragmentation spectra were searched using the SEQUEST HT algorithm against a custom compiled proteome including contaminants using Proteome Discoverer 1.4. Cleavages were allowed after every amino acid, but the search was limited to monoisotopic precursor ions and a peptide mass tolerance of < 10 ppm. Oxidation (+15.995 Da) was set as dynamic modification at methionine residues in all