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 BiochemistryMacquarie University National University of SingaporeSydney, Australia Singapore(shoba.ranganathan@mq.edu.au) (shoba@bic.nus.edu.sg)

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

Stefan Maetschke, U Queensland

MitochondriaEndoplasmic

reticulum

DNADNA

mRNAmRNA

translation

transcription

SP

cTP

mTP

other

Cytosol

CellCell

Nucleus

Legend :

SP : Signal peptide

mTP : mitochondrial targeting peptide

cTP : chloroplast targeting peptide

Secreted protein

Transmembrane protein

Chloroplast

Endo-plasmic reticulum

Polypeptide (Protein)Polypeptide (Protein)

SP

Secreted protein

Protein targeting

4

signal peptide mature protein

cleavage site

N-terminus C-terminus

MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGY...

Signal peptide characteristics

P1-P1’

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 (P7-P6’: 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

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’

P7 P3 P2 P1’ P2’ 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

lipopeptide (blue)

β-lactam (yellow)

DsbA 13-25 (red)

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 enzyme-substrate 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

Signal peptide Mature peptide

small: green; medium: blue; large: red

P3

P1 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 substrate-binding 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 ChooJoo Chuan TongDept. of Biochemistry, Yong Loo Lin

School of Medicine, National University of Singapore

InCoB organizers