Transcript Duplication, Permutation, Adaptation

Modeling Escherichia coli signal peptidase
complex with bound substrate:
Determinants in mature peptide influencing
signal peptide cleavage
Khar Heng Choo & Joo Chuan Tong (I2R)
Shoba Ranganathan
Professor and Chair – Bioinformatics
Adjunct Professor
Biotechnology Research Institute
Dept. of Biochemistry
Macquarie University
National University of Singapore
Sydney, Australia
Singapore
([email protected])
([email protected])
Protein targeting
Discovered by Günter Blobel in the 1970s
and was awarded Nobel Prize in
Physiology or Medicine in 1999 for his
discovery that
“Proteins have intrinsic signals that govern
their transport and localization in the cell”
Protein targeting
Cell
DNA
transcription
Nucleus
mRNA
Transmembrane
protein
Cytosol
Legend :
translation
SP : Signal peptide
Polypeptide (Protein)
SP
mTP
EndoEndoplasmic
Secreted
Secreted
protein
protein
plasmic
reticulum
reticulum
mTP : mitochondrial
targeting peptide
cTP
Mitochondria
cTP : chloroplast
targeting peptide
other
Stefan Maetschke, U Queensland
Chloroplast
Signal peptide characteristics
N-terminus
C-terminus
P1-P1’
MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGY...
mature protein
signal peptide
cleavage
site
4
Introduction
Type I signal peptidases (SPases) are essential
membrane-bound serine proteases responsible
for the cleavage of signal peptides from proteins
that are translocated across biological
membranes – an example of a highly secreted E.
coli protein is the periplasmic dithiol oxidase
(DsbA)
Crystal structure of SPase in complex with signal
peptide not solved
Substrate-binding site and binding specificities
remain poorly understood
Our aims
To develop a structure-based model for E. coli
periplasmic dithiol oxidase (DsbA) 13-25 (P7P6’: H2N-LAFSASAΔAQYEDG-COOH) in
complex with its endogenous type I SPase
To understand how the peptide modeled in this
study could be used to understand the observed
E. coli repertoire of secreted signals, compiled
by our manually curated signal peptide
database.
Data : Sequences & Structures
 107 experimentally validated E. coli type I SPase peptide
substrates extracted from the manually curated signal
peptide database (SPdb) at
http://proline.bic.nus.edu.sg/spdb/
(Choo, Tan and Ranganathan, BMC Bioinformatics
2005, 6:249)
 Redundancy reduction at 80% using CD-HIT
(Li and Godzik, Bioinformatics 2006, 22:1658-1659)
 E. coli type I SPase-bound β-lactam (1B12) and
lipopeptide (1T7D) inhibitors were retrieved from the
Protein Data Bank (PDB)
What do we know?
We have the structures of two inhibitor peptides
at the active site of SPase I: these peptides
mimic the transition state structure of the bound
signal peptide to the signal peptidase.
These inhibitors also occupy positions that are
complementary, with little overlap.
Determinants in the sequence of the mature
protein affect signal peptide processing and
secretion.
Methodology
Thread DsbA P7 to P1' positions against the
solved structures of β-lactam and lipopeptide
inhibitors
P7
P3 P2
P1’ P2’
P6’
Flexible docking using biased Monte Carlo
approach incorporated in ICM (Abagyan et al., J.
Comp. Chem. 1994, 15:488-506) for evaluation
of the electrostatic solvation energy for P2’ to
P6’
Methodology (cont.)
Structures relaxed using ICM software package
conjugate gradient minimization.
In each iteration, new conformations for P2’ to
P6’ were selected based on the Metropolis
criterion with a temperature of 5000K. The
simulation was terminated after 20,000 energy
evaluations.
Intermolecular hydrogen bonds was calculated
using HBPLUS (McDonald and Thornton, J.Mol.
Biol. 1994, 238:777-793).
Superimposition of DsbA 13-25 precursor protein
with lipopeptide and β-lactam inhibitors
DsbA 13-25 (red)
lipopeptide (blue)
β-lactam (yellow)
Results
13 enzyme subsites S7 to S6’ were identified
within the SPase substrate binding site that
might be critical to catalysis
Narrow clefts at S3, S2, S1 and S1’ play direct
roles in the high specificity of the signal peptide
residues
Larger clefts at S3’ and S4’ may be responsible
for the specificity of the mature moieties.
Results
DsbA 13-25 precursor protein is bound to E.
coli type I SPase with a pronounced twist
between positions P3 and P1’
Signal peptide
Mature peptide
Results (cont.)
Our model suggests that the enzymesubstrate contact points extend all the way
from P7 to P6’ of the DsbA precursor
protein.
Other models described earlier only
focused on the P3-P1’ segment and did
not analyze in full the different substrate
binding pockets on either side of the
scissile bond.
Results (cont.)
The S2 subsite has the deepest cavity
can accommodate residues with large
side chains
appears to play an important role in
substrate specificity of E. coli type I
SPase
formerly proposed as the S1 by Paetzel
et al., largely overlaps with the S1
subsite due to a pronounced twist in the
P3 to P1’ binding conformation
Results (cont.)
In contrast to the analysis by Paetzel et
al.(2002), our model reveals that the Ser18 (P2)
side chain is not solvent exposed but is
completely buried.
Disparity between our model and Paetzel et al.
may be attributable to the selection of different
template structures where the structures of the
covalently bound peptide inhibitor complex and
the analogous enzyme LexA were used to guide
the P1 and P3 to P6 positions.
Results (cont.)
The conformation of P3' and P4' allow
their corresponding side-chains to extend
into a large cavity (S3'/S4' subsites)
medium or large residues are preferred
at these two positions for favorable
interactions
medium or large residues both at P3’
(81%) and at P4’ (90%) positions
Sequence logo of AAs size at different positions for
the precursor proteins of 107 experimentally
verified E. coli signal sequences from SPdb
small: green;
medium: blue;
large: red
Signal peptide
Mature peptide
P1
P3
P1’
Results (cont.)
Most residues are tolerated at the +1 (P1’)
position of the mature moiety, with the
exception of the large hydrophobes, Ile,
Met and Trp and Pro, and Arg
Pro is avoided as the rigid positioning of
its backbone hinders docking interactions
with SPase at positions P2’ to P6’
Conclusions
This is the first report on the modeling of a
precursor protein into the entire SPase binding site
Our model provides insights into the binding
conformation of signal peptides and the substratebinding site of E. coli SPase I
SPdb data suggests that signal and mature
moieties should be considered in the development
of predictive tools
Acknowledgements
Khar Heng Choo
Joo Chuan Tong
Dept. of Biochemistry, Yong Loo Lin
School of Medicine, National University of
Singapore
InCoB organizers