A novel mitochondrial and chloroplast peptidasome, PreP

A novel mitochondrial and chloroplast peptidasome, the Presequence Protease (PreP) degrades organellar targeting peptides as well as other unstructured peptides up to 65 amino acid residues in length. PreP belongs to the pitrilysin oligopeptidase family (M16C) containing an inverted zinc-binding motif. The crystal structure of Arabidopsis thaliana PreP, AtPreP, refined at 2.1 ?, revealed a novel mechanism of proteolysis in which two halves of the enzyme connected by a hinge region enclose a large catalytic chamber opening and closing in response to peptide binding. Double knock-out mutant of AtPreP1 and AtPreP2 results in a severe phenotype, including decreased size and growth rate, chlorosis and organellar abnormalities, such as altered chloroplast starch content, partial loss of the integrity of the inner mitochondrial membrane and reduced mitochondrial respiration. PreP homologues are also present in yeast and humans. Interestingly, human PreP has been associated with Alzheimer's disease as it is responsible for degradation of amyloid-β peptide in brain mitochondria.     

Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.     

Domain structure of mitochondrial and chloroplast targeting peptides

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved 'homology blocks' are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides.     

Cleavage-site motifs in mitochondrial targeting peptides

Although mitochondrial targeting peptides lack a common consensus sequence, a certain bias in the positional distribution of amino acids has recently been found. These patterns seem to be associated with cleavage of the precursor proteins by matrix processing proteases. We have extended the previous studies and found new sequence motifs that are conserved within subgroups of mitochondrial targeting peptides. These motifs have certain common themes, indicating that they are associated with cleavage by one single protease. Two of the conserved patterns have a high predictive value, but even for sequences that do not possess these patterns, a fairly accurate prediction of the cleavage site is shown to be possible. We also suggest that a well-conserved RXY decreases (S/A) pattern may be used to engineer efficiently recognized cleavage sites into uncleaved or artificial mitochondrial targeting peptides.     

The action of neutrophil serine proteases on elastin and its precursor

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P₁, which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P₁ in tropoelastin, whereas Lys was not identified at P₁ in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P₂ and P₄′. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.     

Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean

CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biological activity of Medicago truncatula CLE36, which possesses broadly conserved border sequences flanking the putative final active CLE36 peptide product, was assessed. Using in vitro root growth assays and an in vitro root and callus formation assay it is shown that CLE36 peptides of different lengths possess differential biological activities. Using mass spectrometry, Glycine max and Medicago extracellular fluids were each shown to possess an endoproteolytic activity that recognizes and cleaves at border sequences in a synthetic 31 amino acid CLE36 'propeptide bait' to liberate biologically active peptide products. Inhibitor studies suggest that a subtilisin, in combination with a carboxypeptidase, liberated and trimmed CLE36, respectively, to form biologically relevant 11-15 amino acid cleavage products. The 15 amino acid cleavage product is more biologically potent on Arabidopsis than shorter or longer CLE peptides. In situ hybridization shows that the soybean orthologue of CLE36 (GmCLE34) is expressed in the provascular tissue. The results suggest that secreted subtilisins can specifically recognize the border sequences of CLE36 propeptides and liberate biologically active cleavage products. These secreted proteases may affect the stability and biological activity of CLE peptides in the apoplast or be involved in CLE36 processing.     

A truncated analog of a pre-light-harvesting chlorophyll a/b protein II transit peptide inhibits protein import into chloroplasts

It is unclear how transit peptides target nuclear-encoded precursor proteins to the chloroplast. This study establishes the feasibility of using synthetic peptides as competitive inhibitors of chloroplast protein import and as probes for the function of domains within transit peptides. We show that peptide pL(1-20), MAASTMALSSPAFAGKAVNY, an analog of the NH2 terminus of a pre-light harvesting chlorophyll a/b protein II from Arabidopsis, inhibits the import of several Arabidopsis and pea precursor proteins into pea chloroplasts. Inhibition occurs at a step between the initial binding of precursors to the chloroplast and the first proteolytic cleavage event and is not due to interference with ATP availability or chloroplast integrity. Presumably this reflects specific binding of the peptide to the import machinery in the chloroplast envelope. Our data are consistent with the suggestion (Karlin-Neumann, G. A., and Tobin, E. M. (1986) EMBO J. 5, 9-13) that two conserved blocks of amino acids near the NH2-terminus of transit peptides (spanned by peptide pL(1-20] participate in protein targeting. Computer analysis also shows peptide pL(1-20) lacks the amphiphilic properties characteristic of pre-sequences of many nuclear-encoded mitochondrial proteins. This shows a difference in the mechanisms for targeting proteins to chloroplasts and mitochondria.     

Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265, 408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Relative to P1-P1' statine or Phe psi [CH2N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi[CH(OH)CH2]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protease profiling using a fluorescent domino peptide cocktail

Five hexapeptides were prepared containing, in a domino-type arrangement, all 25 possible dipeptides between (1) aromatic, (2) hydrophobic, (3) positively charged, (4) negatively charged, and (5) small and polar amino acids. The peptides were fluorescence labeled at the N-terminus with a (7-coumaryl)oxyacetyl group, allowing the selective detection of N-terminal cleavage products. The five peptides were used as a cocktail reagent in an HPLC analysis. The cocktail produced specific cleavage patterns, or fingerprints, for a variety of proteases. This domino peptide cocktail can be used as a general reagent for protease identification and functional profiling.     

Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP

The dual-targeted mitochondrial and chloroplastic zinc metallooligopeptidase from Arabidopsis, AtPreP, functions as a peptidasome that degrades targeting peptides and other small unstructured peptides. In addition to Zn located in the catalytic site, AtPreP also contains two Mg-binding sites. We have investigated the role of Mg-binding using AtPreP variants, in which one or both sites were rendered unable to bind Mg²⁺. Our results show that metal binding besides that of the active site is crucial for AtPreP proteolysis, particularly the inner site appears essential for normal proteolytic function. This is also supported by its evolutionary conservation among all plant species of PreP.

Structured summary

MINT-7231937, MINT-7232017, MINT-7232035, MINT-7232051, MINT-7232070, MINT-7232090:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) pF1 beta (uniprotkb:P17614) by protease assay (MI:0435)MINT-7232132:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) galanin (uniprotkb:P22466) by protease assay (MI:0435)MINT-7232175:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) Cecropin A (uniprotkb:P14954) by protease assay (MI:0435)MINT-7232163:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) hPrPss (uniprotkb:P04156) by protease assay (MI:0435)     

Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.     

Proteochemometrics analysis of substrate interactions with dengue virus NS3 proteases

The prime side specificity of dengue protease substrates was investigated by use of proteochemometrics, a technology for drug target interaction analysis. A set of 48 internally quenched peptides were designed using statistical molecular design (SMD) and assayed with proteases of four subtypes of dengue virus (DEN-1-4) for Michaelis (K(m)) and cleavage rate constants (k(cat)). The data were subjected to proteochemometrics modeling, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. Detailed analysis of the models then showed that considerably differing physico-chemical properties of amino acids contribute independently to the K(m) and k(cat) activities. For k(cat), only P1' and P2' prime side residues were important, while for K(m) all four prime side residues, P1'-P4', were important. The models could be used to identify amino acids for each P' substrate position that are favorable for, respectively, high substrate affinity and cleavage rate.     

AraPerox. A database of putative Arabidopsis proteins from plant peroxisomes

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.     

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN^E475Q in complex with the products of neurotensin cleavage at 2.7 Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1–40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35–40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.     

Proline residues in the maturation and degradation of peptide hormones and neuropeptides

The proteases involved in the maturation of regulatory peptides like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic amino acid proline. This generates several mature peptides with N-terminal X-Pro-sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In mammals, proline-specific proteases are not involved in the biosynthesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which are then susceptible to broader aminopeptidase attack. The cleavage of several neuropeptides by prolyl endopeptidase has been demonstrated in vitro, but its role in the brain is questionable since the precise localization of the protease is not clarified.     

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.     

Barley serine proteinase inhibitor 2-derived cyclic peptides as potent and selective inhibitors of convertases PC1/3 and furin

Proprotein convertases (PCs) are serine proteases containing a subtilisin-like catalytic domain that are involved in the conversion of hormone precursors into their active form. This study aims at designing small cyclic peptides that would specifically inhibit two members of this family of enzymes, namely, the neuroendocrine PC1/3 and the ubiquitously expressed furin. We studied peptide sequences related to the 18-residue loop identified as the active site of the 83 amino acid barley serine protease inhibitor 2 (BSPI-2). Peptides incorporating mutations at various positions in the sequence were synthesized on solid phase and purified by HPLC. Cyclization was achieved by the introduction of a disulfide bridge between the two Cys residues located at both the N- and C-terminal extremities. Peptides VIIA and VIIB incorporating P4Arg, P2Lys, P1Arg, and P2'Lys were the most potent inhibitors with K(i) around 4 microM for furin and around 0.5 microM for PC1/3. Whereas peptide VIIB behaved as a competitive inhibitor of furin, peptide VIIA acted as a noncompetitive one. However, all peptides were eventually cleaved after variable incubation times by PC1/3 or furin. To avoid this problem, we incorporated at the identified cleavage site a nonscissile aminomethylene bond (psi[CH(2)-NH]). Those pseudopeptides, in particular peptide VIID, were shown not to be cleaved and to inhibit potently furin. Conversely, they were not able to inhibit PC1/3 at all. Those results show the validity of this approach in designing new effective PC inhibitors showing a certain level of discrimination between PC1/3 and furin.     

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6′ in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1′ (with a ~ 32–93% preference for leucine depending on the MMP), and basic and small residues in P2′ and P3′, respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1′ leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1′-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1′. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.