300 uM [U-99% 2H/15N] proMMP-7, 450 uM unlabeled heparin dp8, 93% H2O/7% D2O
deuterated proMMP-7 + heparin dp8
deuterated proMMP-7 + heparin dp8
93% H2O/7% D2O
solution
4
300 uM [U-99% 2H/15N] proMMP-7, 450 uM heparin dp4, 93% H2O/7% D2O
proMMP-7 +/- heparin dp4 The free state of proMMP-7 and its complex with heparin dp4 were each measured without and with the addition of 0.3 mM Gd-EDTA
20 mM imidazole, 10 mM CaCl2, 150 mM NaCl, 20 uM ZnCl2, 10 mM 2-mercaptoethanol
180mM
proMMP-7 + heparin dp8
6.6
1atm
310K
2
20 mM imidazole, 10 mM CaCl2, 150 mM NaCl, 20 uM ZnCl2, 10 mM 2-mercaptoethanol
180mM
proMMP-7 + spin-labeled heparin dp8
6.6
1atm
310K
3
+/- 450 uM hep dp4 and +/- 300 uM GdEDTA 20 mM imidazole, 10 mM CaCl2, 150 mM NaCl, 20 uM ZnCl2, 10 mM 2-mercaptoethanol
180mM
proMMP-7 +/- hep dp4 +/- GdEDTA
6.6
1atm
310K
4
Conditions used for assigning the peaks and determining the solution structure (PDB ID: 2MZE) 20 mM imidazole, 10 mM CaCl2, 20 uM ZnCl2, 10 mM 2-mercaptoethanol
30mM
proMMP-7, free state
6.6
1atm
310K
-
NMR measurement
NMR spectrometer
Type: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 800 MHz / Details: TCI cryoprobe
-
Processing
Software
Name
Version
Classification
CYANA
2.1
refinement
SYBYL-X
2.1.1
refinement
NMR software
Name
Version
Developer
Classification
CYANA
2.1
Guntert, MumenthalerandWuthrich
refinement
SYBYL-X
2.1.1
Certara (Tripos)
refinement
Sparky
Goddard
dataanalysis
CcpNMR
CCPN
chemicalshiftassignment
TopSpin
3.1
BrukerBiospin
processing
CYANA
2.1
Guntert, MumenthalerandWuthrich
structurecalculation
Refinement
Method: torsion angle dynamics / Software ordinal: 1 Details: Explicit distance restraints to the TEMPO spin-labeled reducing end of heparin dp8 were obtained from the PREs (T2) using an equation from Battiste and Wagner (2000, Biochemistry) simplified ...Details: Explicit distance restraints to the TEMPO spin-labeled reducing end of heparin dp8 were obtained from the PREs (T2) using an equation from Battiste and Wagner (2000, Biochemistry) simplified by WHTC greater than1 to the form r equal to 4KTC/T2 given by Koppisetti et al. (2014, Nature Comms.). The rotational correlation time TC of the protein-heparin complexes was obtained from amide 15N cross-correlation rates nxy (Liu and Prestegard, 2008, J. Magn. Reson.) interpreted by the spectral density expression used in the TRACT approach (Lee et al., 2006. J. Magn. Reson.). Upper bounds were set at 15% above the distance estimate. Lower bounds were implicitly at van der Waals distance.Ambiguous distance restraints from any residue of heparin dp8 to explicit protein amide groups were applied on the basis of amide chemical shift perturbations from heparin dp8 or protection of amides by heparin dp4 from line broadening by Gd.EDTA with deltaT2 greater than 37/sec. Ambiguous distance restraints from any residue of heparin dp8 to lysine amino or arginine ureido groups were also applied on the basis of mutations that impaired activation of proMMP-7 by heparin dp16. The combination of the intermolecular distance restraints from the explicit PRE and NOE measurements and the ambiguous sources were used to dock coordinates of heparin dp8 with the NMR solution structure of human proMMP-7 of Prior et al. (2015, Structure). To enable molecular flexibility widely through the proMMP-7 and heparin dp8 chains while maintaining the structural integrity of the proMMP-7 during the restrained docking simulations, CYANA 2.1 (Guntert and Buchner, 2015, J. Biomol. NMR) was utilized in concert with the intramolecular proMMP-7 NOE-derived distance restraints and chemical shift-derived dihedral restraints of the NMR structure (Prior et al., 2015, Structure). CYANA topology files describing the sugar monomers were derived from topology files curated by the Automated Topology Builder (Malde et al., 2011, J. Chem. Theory Comput.) and based on accession number 9804 for 2-O-sulfo-alpha-L-idopyranuronic acid (IDS or IdoA2S) and 9778 for N,O6-disulfo-glucosamine (SGN or GlcNS6S). The globally flexible docking simulations required the proMMP-7 polypeptide to be linked to the calcium and zinc ions and heparin dp8 chains via tethers of non-interacting pseudoatoms. With this tethering and flexible structural integrity enforced, the carbohydrate chains were docked with the intermolecular distance restraints. Energy minimization was performed using SYBYL-X
NMR representative
Selection criteria: closest to the average
NMR ensemble
Conformer selection criteria: structures with the least restraint violations Conformers calculated total number: 120 / Conformers submitted total number: 16
+
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