PDBsum entry 2vh5

Immune system

PDB id: 2vh5

Contents

Protein chains

114 a.a. *

104 a.a. *

166 a.a. *

Ligands

GTP

Metals

_ZN ×3

_MG

Waters ×42

* Residue conservation analysis

PDB id:

2vh5

Name:

Immune system

Title:

Crystal structure of hras(g12v) - anti-ras fv (disulfide free mutant) complex

Structure:

Anti-ras fv heavy chain. Chain: h. Engineered: yes. Anti-ras fv light chain. Chain: l. Engineered: yes. Gtpase hras. Chain: r. Fragment: residues 1-166.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Expression_system_variant: c41.

Resolution:

2.70Å R-factor: 0.215 R-free: 0.291

Authors:

T.Tanaka,R.L.Williams,T.H.Rabbitts

Key ref:

T.Tanaka and T.H.Rabbitts (2008). Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds. J Mol Biol, 376, 749-757. PubMed id: 18187153 DOI: 10.1016/j.jmb.2007.11.085

Date:

19-Nov-07 Release date: 22-Jan-08

Protein chain

No UniProt id for this chain

Struc: 114 a.a.

Protein chain

No UniProt id for this chain

Struc: 104 a.a.

Protein chain

P01112  (RASH_HUMAN) -  GTPase HRas from Homo sapiens

Seq: 189 a.a.

Struc: 166 a.a.*

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

Enzyme reactions

• Enzyme class: Chain R: E.C.3.6.5.2 - small monomeric GTPase.
• Reaction: GTP + H2O = GDP + phosphate + H⁺

GTP (Bound ligand (Het Group name = GTP) corresponds exactly) + H2O = GDP + phosphate + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2007.11.085

J Mol Biol 376:749-757 (2008)

PubMed id: 18187153

Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds.

T.Tanaka, T.H.Rabbitts.

ABSTRACT

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-A crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.

Selected figure(s)

Figure 3.
Fig. 3. Structural comparison of native and disulfide-free RAS–anti-RAS Fv complex. (a) Representation of HRAS bound by disulfide-free Fv. HRAS(G12V) (green) is shown as a molecular surface model. The switch I and switch II regions of RAS are shown in cyan and magenta, respectively, and guanidine triphosphate (GTP) is shown in orange. The Fv [comprising VH (blue) and VL (red)] is shown as a ribbon representation, with the CDRs of VH and VL in blue and pink, respectively. The positions 23 and 104 where cysteines were substituted by alanine and valine in VH, or by valine and alanine in VL, are in yellow. (b) Superimposition of anti-RAS Fv native form^2 and disulfide-free Fv with alanine–valine substitutions in VH [VH#6(AV)] and with valine–alanine substitutions in VL(VA). The structure is shown as a stereo view of the Cα trace VH and VL of the native forms shown in cyan and pink and of the disulfide-free forms shown in blue and red, respectively. (c and d) 2F[o] − F[c] electron density maps (contoured at 0.5σ) around the disulfide bond regions of VH (c) and VL (d): native form (left) and disulfide-free mutant (right). The distance of Cα atom between the residues of cysteines or their substitutions is shown as a dotted line in yellow.

Figure 4.
Fig. 4. Electron density maps of VH and VL CDRs. Views of VH and VL CDR structures with a 2F[o] − F[c] electron density map. The map is contoured at 0.5σ. Left panels show the native anti-RAS VH (a–c) and VL (d–f) single domains. Right panels show the disulfide-free form. (a) VHCDR1; (b) VHCDR2; (c) VHCDR3; (d) VLCDR1; (e) VLCDR2; (f) VLCDR3.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 376, 749-757) copyright 2008.

Figures were selected by an automated process.