title: 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.
abstract: 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.
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Eckhard
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Germany.
authors.forename: Pitter F
authors.initials: PF
authors.lastname: Huesgen
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Swissmedic, Swiss Agency for Therapeutic Products, Bern, Switzerland.
authors.forename: Caroline L
authors.initials: CL
authors.lastname: Bellac
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Georgina S
authors.initials: GS
authors.lastname: Butler
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Inception Sciences, Vancouver, BC, Canada.
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Cox
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Antoine
authors.initials: A
authors.lastname: Dufour
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Verena
authors.initials: V
authors.lastname: Goebeler
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Reinhild
authors.initials: R
authors.lastname: Kappelhoff
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.
authors.forename: Ulrich Auf dem
authors.initials: UAD
authors.lastname: Keller
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Theo
authors.initials: T
authors.lastname: Klein
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Department of Pathology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Philipp F
authors.initials: PF
authors.lastname: Lange
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Giada
authors.initials: G
authors.lastname: Marino
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Charlotte J
authors.initials: CJ
authors.lastname: Morrison
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Anna
authors.initials: A
authors.lastname: Prudova
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Department of Bioquímica y Biología Molecular, Universidad de Oviedo, Oviedo, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Rodriguez
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Present address: Ottawa Institute of Systems Biology, University of Ottawa, Canada.
authors.forename: Amanda E
authors.initials: AE
authors.lastname: Starr
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Yili
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada. Electronic address: chris.overall@ubc.ca.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D006801:Humans; D020782:Matrix Metalloproteinases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D019151:Peptide Library; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D019151:Peptide Library; D020782:Matrix Metalloproteinases
keywords: MMPs; PICS; Peptide docking; Proteomics; Quenched-fluorescent peptides; Specificity profiling