Barrel-shaped ClpP Proteases Display Attenuated Cleavage Specificities

ClpP is a self-compartmentalizing protease with crucial roles in bacterial and mitochondrial protein quality control. Although the ClpP homocomplex is composed of 14 equivalent active sites, it degrades a multitude of substrates to small peptides, demonstrating its capability to carry out diverse cleavage reactions. Here, we show that ClpP proteases from <i>E. coli</i>, <i>S. aureus</i>, and human mitochondria exhibit preferences for certain amino acids in the P1, P2, and P3 positions using a tailored fluorogenic substrate library. However, this high specificity is not retained during proteolysis of endogenous substrates as shown by mass spectrometric analysis of peptides produced in ClpXP-mediated degradation reactions. Our data suggest a mechanism that implicates the barrel-shaped architecture of ClpP not only in shielding the active sites to prevent uncontrolled proteolysis but also in providing high local substrate concentrations to enable efficient proteolytic processing. Furthermore, we introduce customized fluorogenic substrates with unnatural amino acids that greatly surpass the sensitivity of previously used tools. We used these to profile the activity of cancer-patient- and Perrault-syndrome-derived ClpP mutant proteins

Visualization of PK401 with purified NSP4 and all NSP’s.

<p>(A) NSP4 was treated with PK401 in a range from 1 to 2000nM. (B) 100nM of NE, PR3, CatG and NSP4 with or without 100nM of PK401. (A, B) Samples were denatured in SDS sample buffer, run in SDS/PAGE followed by membrane transfer. The blot was developed with fluorescently-tagged streptavidin and imaged by fluorescence scanning (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132818#pone.0132818.s001" target="_blank">S1 Text</a>).</p

Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4

<div><p>Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.</p></div

Scheme of the HyCoSuL P1 Arg library.

<p>The general library structure contains tetrapeptide derivatives with the sequence Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents one of 120 fixed natural or unnatural amino acids and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met) with ACC (7-amino-4-carbamoylmethylcoumarin) as a reporter group.</p

Kinetic analysis of tetrapeptide substrates for NSP4.

<p>Results are shown as an average of a minimum of 2 separate experiments with S.D.</p

Structures of the optimized NSP4 substrates based on natural (PK417 and PK418) and natural/unnatural amino acids (PK421 and PK431).

<p>The activity-based probe (PK401), a diphenyl phosphonate derived from the optimal substrate sequence—PK421—is shown as the last structure.</p

Kinetic parameters/constants for the hydrolysis of Ac-hCha-Phe(guan)-Oic-Arg-ACC substrate by neutrophil serine proteases to three significant digits.

<p>NA–no activity detected.</p><p>Kinetic parameters/constants for the hydrolysis of Ac-hCha-Phe(guan)-Oic-Arg-ACC substrate by neutrophil serine proteases to three significant digits.</p

Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg^P(OPh)₂ (PK401).

<p>NI–no inhibition observed; K_m values relate to the substrate used for analysis,</p><p>* K_m for this substrate was above 100μM, the concentration used in the assay. AMC – 7-amino-4-methylcoumarin.</p><p>Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg^P(OPh)₂ (PK401).</p

Determination of NSP4 substrate specificity.

<p>Preferences in the P4-P2 positions were determined by screening HyCoSuL, which contains tetramer peptides with the general structures Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents fixed natural or unnatural amino acid and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met). Screening was performed on a SpectraMax Gemini plate reader. Substrate hydrolysis rates were normalized to the most active component (100%) y axis. Natural amino acids are colored grey, unnatural black. Results are shown as an average of 3 experiments with S.D.</p

Preferred natural amino acids substrates for recombinant <i>Pf</i>M1AAP and <i>Pf</i>M17LAP.

<p>Initial screening of the 19-membered natural amino acid library. Enzyme activity was monitored using an fmax multi-well fluorescence plate reader (Molecular Devices) at excitation wavelength of 355 nm and an emission wavelength of 460 nm. he x-axis represents the abbreviated amino acid names (for full name and structure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031938#pone.0031938.s001" target="_blank">Figure S1</a>). The y-axis represents the average relative activity expressed as a percent of the best amino acid. In the heat map view the most preferred positions are displayed in bright red, whereas a complete lack of activity is in black, with intermediate values represented by intermediate shades of red.</p