Crystal structure of SecB from Escherichia coli

doi:10.1016/j.jsb.2003.09.012

Journal of Structural Biology

Volume 144, Issue 3, December 2003, Pages 313-319

https://doi.org/10.1016/j.jsb.2003.09.012 Get rights and content

Abstract

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 Å resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.

Introduction

As protein synthesis in Escherichia coli takes place in the cytosol, export is essential for proteins functioning outside the cytosol. Early steps in this export process involve the translocation of precursor proteins across the inner membrane. This is accomplished by the translocation machinery, a well studied complex of Sec-proteins (for a review see (Driessen et al., 2001)). The integral membrane proteins SecY, SecE, and SecG form together with the peripherally bound ATPase SecA the translocase. SecA is able to insert and deinsert into the membrane upon binding and hydrolysis of ATP, providing the energy to drive the translocation process (Driessen et al., 1995). SecA has also been found in the cytosol and together with SecB it forms the soluble part of the translocation machinery (Hartl et al., 1990). SecB is a molecular chaperone that binds to a subset of precursor proteins, thereby preventing them from aggregating (Kumamoto, 1990). The affinity of SecB for the C-terminus of SecA plays an important role in the correct targeting of the precursor protein to the translocase (Breukink et al., 1995). SecB is active as a tetramer and is highly selective towards its substrates. SecB substrates share no sequence homology, but a minimal binding motif consists of nine residues of charged and aromatic amino acids (Knoblauch et al., 1999). Although it is the signal sequence that is an earmark for export proteins, SecB does not exploit this as a binding motif but mainly binds to regions in the mature part of precursor proteins (Topping and Randall, 1994). Tight binding of SecB to a substrate is likely to be modulated by the ability of the chaperone to interact at multiple sites of a single substrate (Randall et al., 1998).

Previously, the crystal structure of SecB from Haemophilus Influenzae was determined to 2.5 Å resolution (Xu et al., 2000). Here we report the three-dimensional crystal structure of SecB from E. coli at 2.35 Å resolution. Together with a wealth of biochemical data that is available for this E. coli chaperone, the structure allows us to speculate on the mechanism by which SecB binds and releases precursor proteins and interacts with SecA.

Section snippets

Crystallisation and data collection

SecB was crystallised as described previously (Dekker et al., 1999). Diffraction data were collected from a single crystal at EMBL beamline X11 (DESY, Hamburg). A V_matthews of 2.6 A³/Da suggests one tetramer per asymmetric unit, with a solvent content of 53.2%. Data were processed with DENZO and scaled with SCALEPACK (Otwinowski and Minor, 1997). Table 1 summarises data collection and processing statistics.

Structure solution and refinement

The structure was solved by molecular replacement with AmoRe (Navaza, 1994), using as a

Structure description

The crystal structure of SecB from E. coli was solved by molecular replacement using the co-ordinates of SecB from H. Influenzae (Xu et al., 2000), which has 55% sequence identity with SecB from E. coli. Fig. 1 shows the Cα backbone of the chaperone. SecB is a 155-residue protein that forms a tetramer with a molecular mass of 68.8 kDa. The tetramer is build up as a dimer of dimers. Each monomer named A to D, folds into a 4-stranded Greek key anti-parallel β-sheet with a long α-helix packed

Conclusion

The chaperone SecB from E. coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. SecB is involved in two fundamentally different types of interactions: interactions with a diverse set of unfolded proteins, that are in rapid equilibrium with the free pool (Topping and Randall, 1997) and the unique interaction with SecA that makes the precursor enter the translocation pathway. SecB might also engage in functions completely unrelated to

Acknowledgments

We are grateful to Clasien Oomen for her contribution. We thank V.S. Lamzin and M. Wilmanns for data collection at the EMBL at DESY, Hamburg. Support was provided under the EU TMR program to the EMBL Hamburg Outstation, Reference ERB4062PL970104. This work was supported in part by the Council of Chemical Sciences of the Netherlands Organisation for Scientific Research (NWO-CW). CD received support from the Commission of the European Communities (Grant QLK2-CT-1999-01293).

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Crystal structure of SecB from Escherichia coli

Abstract

Introduction

Section snippets

Crystallisation and data collection

Structure solution and refinement

Structure description

Conclusion

Acknowledgments

J. Biol. Chem.

J. Struct. Biol.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

TIBS

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Crystallography & NMR system: a new software suite for macromolecular structure determination

Acta Crystallogr. D