4OEM

Crystal structure of Cathepsin C in complex with dipeptide substrates


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.52 Å
  • R-Value Free: 0.178 
  • R-Value Work: 0.164 
  • R-Value Observed: 0.165 

wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.1 of the entry. See complete history


Literature

The amino-acid substituents of dipeptide substrates of cathepsin C can determine the rate-limiting steps of catalysis.

Rubach, J.K.Cui, G.Schneck, J.L.Taylor, A.N.Zhao, B.Smallwood, A.Nevins, N.Wisnoski, D.Thrall, S.H.Meek, T.D.

(2012) Biochemistry 51: 7551-7568

  • DOI: https://doi.org/10.1021/bi300719b
  • Primary Citation of Related Structures:  
    4OEL, 4OEM

  • PubMed Abstract: 

    We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k(cat)/K(a) and k(cat) values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k(cat)/K(a) (1.6 ± 0.09 μM(-1) s(-1)) and k(cat) (255 ± 6 s(-1)), an inverse sKIE on k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.6 ± 0.15), a modest, normal sKIE on k(cat) ((D)k(cat) = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s(-1)). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k(ac)) and deacylation (k(dac)) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697-8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k(cat)/K(a) values of 0.49 ± 0.07 μM(-1 )s(-1) and 5.3 ± 0.5 μM(-1 )s(-1), and k(cat) values of 28 ± 1 s(-1) and 25 ± 0.5 s(-1). Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.74 ± 0.05) and normal for k(cat) ((D)k(cat) = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s(-1) and 390 s(-1), respectively), from which it was determined that k(ac) ≫ k(dac) ∼ k(cat). The third substrate type, Gly-Ile-AMC, gave very low values of k(cat)/K(a) (0.0015 ± 0.0001 μM(-1) s(-1)) and k(cat) (0.33 ± 0.02 s(-1)), no sKIEs, ((D)(k(cat)/K(a)) = 1.05 ± 0.5 and (D)k(cat) = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which k(dac) > k(ac) ∼ k(cat), and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P(1) position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases.


  • Organizational Affiliation

    Department of Biological Reagents and Assay Development, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Dipeptidyl peptidase 1 Heavy chain370Homo sapiensMutation(s): 1 
Gene Names: CPPICTSC
EC: 3.4.14.1
UniProt & NIH Common Fund Data Resources
Find proteins for P53634 (Homo sapiens)
Explore P53634 
Go to UniProtKB:  P53634
PHAROS:  P53634
GTEx:  ENSG00000109861 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP53634
Sequence Annotations
Expand
  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
Dipeptidyl peptidase 1 Light chain70Homo sapiensMutation(s): 0 
Gene Names: CTSCCPPI
EC: 3.4.14.1
UniProt & NIH Common Fund Data Resources
Find proteins for P53634 (Homo sapiens)
Explore P53634 
Go to UniProtKB:  P53634
PHAROS:  P53634
GTEx:  ENSG00000109861 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP53634
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 6 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NAG
Query on NAG

Download Ideal Coordinates CCD File 
F [auth A],
G [auth A],
H [auth A]
2-acetamido-2-deoxy-beta-D-glucopyranose
C8 H15 N O6
OVRNDRQMDRJTHS-FMDGEEDCSA-N
LYS
Query on LYS

Download Ideal Coordinates CCD File 
D [auth A]LYSINE
C6 H15 N2 O2
KDXKERNSBIXSRK-YFKPBYRVSA-O
SO4
Query on SO4

Download Ideal Coordinates CCD File 
E [auth A]SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
GLY
Query on GLY

Download Ideal Coordinates CCD File 
C [auth A]GLYCINE
C2 H5 N O2
DHMQDGOQFOQNFH-UHFFFAOYSA-N
EDO
Query on EDO

Download Ideal Coordinates CCD File 
J [auth A],
K [auth A],
L [auth A],
M [auth A]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
I [auth A]CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.52 Å
  • R-Value Free: 0.178 
  • R-Value Work: 0.164 
  • R-Value Observed: 0.165 
  • Space Group: I 2 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 84.92α = 90
b = 89.016β = 90
c = 115.098γ = 90
Software Package:
Software NamePurpose
BUSTER-TNTrefinement
PDB_EXTRACTdata extraction
HKL-2000data reduction
SCALEPACKdata scaling
AMoREphasing
BUSTERrefinement

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2015-03-25
    Type: Initial release
  • Version 1.1: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Data collection, Database references, Derived calculations, Structure summary