PDBsum entry 1twb

Hydrolase

PDB id: 1twb

Contents

Protein chains

106 a.a. *

Ligands

ALA-CYS-ASN-ASP-GLU-ASN-TYR-ALA ×2

Waters ×90

* Residue conservation analysis

PDB id:

1twb

Name:

Hydrolase

Title:

Sspb disulfide crosslinked to an ssra degradation tag

Structure:

Stringent starvation protein b homolog. Chain: a, b. Fragment: substrate binding domain. Synonym: sspb. Engineered: yes. Mutation: yes. Ssra peptide. Chain: c, d. Fragment: protease recognition tag.

Source:

Haemophilus influenzae. Organism_taxid: 727. Gene: sspb, hi1440. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes

Biol. unit:

Dimer (from PQS)

Resolution:

1.90Å R-factor: 0.235 R-free: 0.268

Authors:

D.N.Bolon,R.A.Grant,T.A.Baker,R.T.Sauer

Key ref:

D.N.Bolon et al. (2004). Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease. Mol Cell, 16, 343-350. PubMed id: 15525508 DOI: 10.1016/j.molcel.2004.10.001

Date:

30-Jun-04 Release date: 16-Nov-04

Protein chains

P45206  (SSPB_HAEIN) -  Stringent starvation protein B homolog from Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)

Seq: 150 a.a.

Struc: 106 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1016/j.molcel.2004.10.001

Mol Cell 16:343-350 (2004)

PubMed id: 15525508

Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease.

D.N.Bolon, R.A.Grant, T.A.Baker, R.T.Sauer.

ABSTRACT

The SspB adaptor enhances ClpXP degradation by binding the ssrA degradation tag of substrates and the AAA+ ClpX unfoldase. To probe the mechanism of substrate delivery, we engineered a disulfide bond between the ssrA tag and SspB and demonstrated otherwise normal interactions by solving the crystal structure. Although the covalent link prevents adaptor.substrate dissociation, ClpXP degraded GFP-ssrA that was disulfide bonded to the adaptor. Thus, crosslinked substrate must be handed directly from SspB to ClpX. The ssrA tag in the covalent adaptor complex interacted with ClpX.ATPgammaS but not ClpX.ADP, suggesting that handoff occurs in the ATP bound enzyme. By contrast, SspB alone bound ClpX in both nucleotide states. Similar handoff mechanisms will undoubtedly be used by many AAA+ adaptors and enzymes, allowing assembly of delivery complexes in either nucleotide state, engagement of the recognition tag in the ATP state, and application of an unfolding force to the attached protein following hydrolysis.

Selected figure(s)

Figure 1.
Figure 1. Structure of the Covalent Adaptor-Substrate Complex(A) Aligned structures of the disulfide-linked SspB-SBD^S-SssrA complex (pink/red) with the noncovalent wild-type complex (cyan/blue; PDB code 1OU8) (Levchenko et al., 2003). SspB is displayed in ribbon representation, and the main chain atoms of the ssrA peptide are in stick representation. The disulfide bond (yellow) is displayed in CPK representation.(B) Simulated-annealing omit map of electron density for a portion of the ssrA peptide and the disulfide crosslink to SspB. The ssrA peptide and Cys44 of SspB were omitted during map calculations. The 1.9 Å 2F[0] − F[C] map is contoured at 0.7σ.(C) Sequence of the ssrA tag showing sites of contact with SspB or ClpX and the mutant cysteine used for crosslinking to SspB.(D) Surface/stick representation of one subunit of the covalent SspB-SBD^S-SssrA complex. Tag residues 9–11 (shown in purple) were not visible in the crystal structure and were modeled in an energetically favorable conformation for this figure.

Figure 3.
Figure 3. ClpX Binding Assayed by Fluorescence Anisotropy of Fluorescein-Labeled SspB VariantsExperiments were performed using 25 nM concentrations of the fluorescein-labeled proteins at 30°C in PD200 buffer with 5 mM ATPγS (upper panel) or 5 mM ADP (lower panel). Competition experiments contained ClpX[6] (125 nM in ATPγS experiments; 4 μM in ADP experiments) and ^FLSspB^S-SssrA (25 nM), in the presence or absence of competitors (500 μM XB peptide; 500 μM ssrA peptide).

The above figures are reprinted by permission from Cell Press: Mol Cell (2004, 16, 343-350) copyright 2004.

Figures were selected by an automated process.