PDBsum entry 2g60

Immune system

PDB id: 2g60

Contents

Protein chains

213 a.a. *

208 a.a. *

Waters ×384

* Residue conservation analysis

PDB id:

2g60

Name:

Immune system

Title:

Structure of anti-flag m2 fab domain

Structure:

Anti-flag m2 fab light chain. Chain: l. Anti-flag m2 fab heavy chain. Chain: h

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Organism_taxid: 10090

Biol. unit:

Dimer (from PQS)

Resolution:

1.85Å R-factor: 0.235 R-free: 0.278

Authors:

T.P.Roosild

Key ref:

T.P.Roosild et al. (2006). Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins. Acta Crystallograph Sect F Struct Biol Cryst Commun, 62, 835-839. PubMed id: 16946459 DOI: 10.1107/S1744309106029125

Date:

23-Feb-06 Release date: 12-Sep-06

Protein chain

Q65ZC0  (Q65ZC0_MOUSE) -  Kappa light chain C_region (Fragment) from Mus musculus

Seq: 219 a.a.

Struc: 213 a.a.*

Protein chain

Q5BJZ2  (Q5BJZ2_RAT) -  LOC367586 protein from Rattus norvegicus

Seq: 458 a.a.

Struc: 208 a.a.*

* PDB and UniProt seqs differ at 68 residue positions (black crosses)

DOI no: 10.1107/S1744309106029125

Acta Crystallograph Sect F Struct Biol Cryst Commun 62:835-839 (2006)

PubMed id: 16946459

Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins.

T.P.Roosild, S.Castronovo, S.Choe.

ABSTRACT

The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ;soluble' portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K(+) channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 A resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.

Selected figure(s)

Figure 1.
Figure 1 Anti-FLAG antibody binding to the K^+ channel KvPae promotes its detergent-solubilized stability. (a) Topology of KvPae with six transmembrane segments (S1-S6) and a re-entrant loop forming the K^+ ion-selectivity filter (P) showing the location of the inserted FLAG epitope. The ten amino acids shown replaced a single glycine (residue 54) in the native protein. (b) Gel-filtration analysis of LDAO-solubilized KvPae (32 kDa) reveals a spectrum of oligomers ranging in size from tetrameric to aggregates up to the 5 MDa void volume of the column (red). In contrast, the KvPae-Fab M2 complex elutes as a symmetrical monodisperse peak of the expected size [(32 kDa KvPae + 50 kDa Fab) × 4 (i.e. homotetramer) + 150 kDa detergent micelle = 478 kDa] (blue).

The above figure is reprinted by permission from the IUCr: Acta Crystallograph Sect F Struct Biol Cryst Commun (2006, 62, 835-839) copyright 2006.

Figure was selected by an automated process.