PDBsum entry 2c4i

Glycoprotein

PDB id: 2c4i

Contents

Protein chain

242 a.a.

Ligands

BTN ×2

SO4

Waters ×118

PDB id:

2c4i

Name:

Glycoprotein

Title:

Crystal structure of engineered avidin

Structure:

Avidin. Chain: a. Engineered: yes

Source:

Gallus gallus. Chicken. Organism_taxid: 9031. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PDB file)

Resolution:

1.95Å R-factor: 0.200 R-free: 0.246

Authors:

V.P.Hytonen,J.Horha,T.T.Airenne,E.A.Niskanen,K.Helttunen,M.S.Johnson, T.A.Salminen,M.S.Kulomaa,H.R.Nordlund

Key ref:

V.P.Hytönen et al. (2006). Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin. J Mol Biol, 359, 1352-1363. PubMed id: 16787776 DOI: 10.1016/j.jmb.2006.04.044

Date:

19-Oct-05 Release date: 05-Jul-06

Protein chain

P02701  (AVID_CHICK) -  Avidin from Gallus gallus

Seq: 152 a.a.

Struc: 242 a.a.*

* PDB and UniProt seqs differ at 31 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2006.04.044

J Mol Biol 359:1352-1363 (2006)

PubMed id: 16787776

Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.

V.P.Hytönen, J.Hörhä, T.T.Airenne, E.A.Niskanen, K.J.Helttunen, M.S.Johnson, T.A.Salminen, M.S.Kulomaa, H.R.Nordlund.

ABSTRACT

Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilised the protein. The modified dcAvd forms were found to retain their characteristic pseudotetrameric state both at high and low pH, and were shown to bind D-biotin at levels comparable to that of wild-type chicken avidin. The crystal structure of dcAvd-biotin complex at 1.95 Angstroms resolution demonstrates the formation of the functional dcAvd pseudotetramer at the atomic level and reveals the molecular basis for its special properties. Altogether, our data facilitate further engineering of the biotechnologically valuable dcAvd scaffold and gives insights into how to guide the quaternary structure assembly of oligomeric proteins.

Selected figure(s)

Figure 1.
Figure 1. Possible quaternary assemblies of dcAvd. (a) Schematic presentation of the possible quaternary structure configurations of dcAvd pseudotetramers. Domains equal to cpAvd5→4 and cpAvd6→5 of dcAvd are coloured red and blue, respectively. Based on SDS-PAGE analysis (Figure 2), the dcAvd(I117C[5→4]), dcAvd(I117C[5→4]M96H[6→5]) and dcAvd(I117C[5→4]I117H[6→5]) mutants are a mixture of two conformations (double-headed arrow) but the dcAvd(I117C[5→4]V115H[6→5] mutant did form a single, disulfide bridge locked conformation (unequal double-headed arrow). The configuration of the quaternary structure could not be determined for dcAvd(I117C[5→4]I117C[6→5]) mutant and for the wt dcAvd in our experimental setup (indicated with question mark). The quaternary assembly observed in the X-ray structure of dcAvd is shown inside a black square and the engineered disulfide locked assembly, dcAvd(I117C[5→4]V115H[6→5]), inside a green square. Mutations are shown by symbols: cysteine, bar; histidine, pentagon; isoleucine, methionine and valine, oval. (b) View of the 1-3 subunit interface from the X-ray structure of dcAvd illustrating the symmetry and hydrophobic character of the interface. Colouring as in (a). The side-chains of residues 96, 115 and 117 are shown as stick models. Both V115 and I117 are located on strand β8, whereas M96 is located on strand β7. Structural water molecules within 4 Å of residues in the interface are shown as dotted spheres. The four valine residues at position 115 (boxed) form a hydrophobic core shielded from solvent and are positioned at the intersection of the 1-2, 1-3 and 1-4 subunit interfaces. (c) The view is rotated −90° around the y-axis with respect to (b). Figure 1. Possible quaternary assemblies of dcAvd. (a) Schematic presentation of the possible quaternary structure configurations of dcAvd pseudotetramers. Domains equal to cpAvd5→4 and cpAvd6→5 of dcAvd are coloured red and blue, respectively. Based on SDS-PAGE analysis ([3]Figure 2), the dcAvd(I117C[5→4]), dcAvd(I117C[5→4]M96H[6→5]) and dcAvd(I117C[5→4]I117H[6→5]) mutants are a mixture of two conformations (double-headed arrow) but the dcAvd(I117C[5→4]V115H[6→5] mutant did form a single, disulfide bridge locked conformation (unequal double-headed arrow). The configuration of the quaternary structure could not be determined for dcAvd(I117C[5→4]I117C[6→5]) mutant and for the wt dcAvd in our experimental setup (indicated with question mark). The quaternary assembly observed in the X-ray structure of dcAvd is shown inside a black square and the engineered disulfide locked assembly, dcAvd(I117C[5→4]V115H[6→5]), inside a green square. Mutations are shown by symbols: cysteine, bar; histidine, pentagon; isoleucine, methionine and valine, oval. (b) View of the 1-3 subunit interface from the X-ray structure of dcAvd illustrating the symmetry and hydrophobic character of the interface. Colouring as in (a). The side-chains of residues 96, 115 and 117 are shown as stick models. Both V115 and I117 are located on strand β8, whereas M96 is located on strand β7. Structural water molecules within 4 Å of residues in the interface are shown as dotted spheres. The four valine residues at position 115 (boxed) form a hydrophobic core shielded from solvent and are positioned at the intersection of the 1-2, 1-3 and 1-4 subunit interfaces. (c) The view is rotated −90° around the y-axis with respect to (b).

Figure 3.
Figure 3. Stereo representations of the X-ray structure of dcAvd. (a) The functional unit of dcAvd. The monomer of the asymmetric unit (I) and its crystallographic symmetry mate (II) were used to create the pseudotetramer. Domains equal to cpAvd5→4 and cpAvd6→5 of dcAvd are coloured red and blue, respectively. The positions of the linker regions L1-L3 (see the text for details) are indicated (* represents L2). The pseudosubunits 1 to 4 of the dcAvd structure are indicated by circled numbers as described by Livnah et al. (3). The bound biotin molecules are shown as spheres. (b) A monomer of dcAvd. The view is rotated −90° around the y-axis with respect to (a). Colouring and labelling as in (a). The N and C terminus of dcAvd are indicated. (c) Superimposition of the cpAvd5→4 domain with the cpAvd6→5 domain, and with the A subunits of two known avidin−biotin complex structures (PDB code 2AVI (light grey) and 1AVD (grey)). The N and C termini of the native avidin structures are labelled with an asterisk. Other labels and colouring are as in (b). Only the biotin of cpAvd5→4 is shown. (d) Comparison of the biotin binding modes of the cpAvd5→4 domain, cpAvd6→5 domain, 2AVI and 1AVD structures. The residues are labelled according to wt avidin. The label for residue Phe72, which is in a clearly different conformation in the cpAvd6→5 domain compared to the other shown structures, is indicated with a yellow background. The weighted difference F[o]−F[c] electron density map (cyan), calculated in the absence of biotin, is drawn with a 2.2 Å radius around the atoms of D-biotin of the final structure of the cpAvd5→4 domain. Only side-chains of amino acids are shown except for residues 37−40 of the loop between β-strands three and four. Figure 3. Stereo representations of the X-ray structure of dcAvd. (a) The functional unit of dcAvd. The monomer of the asymmetric unit (I) and its crystallographic symmetry mate (II) were used to create the pseudotetramer. Domains equal to cpAvd5→4 and cpAvd6→5 of dcAvd are coloured red and blue, respectively. The positions of the linker regions L1-L3 (see the text for details) are indicated (* represents L2). The pseudosubunits 1 to 4 of the dcAvd structure are indicated by circled numbers as described by Livnah et al. (3). The bound biotin molecules are shown as spheres. (b) A monomer of dcAvd. The view is rotated −90° around the y-axis with respect to (a). Colouring and labelling as in (a). The N and C terminus of dcAvd are indicated. (c) Superimposition of the cpAvd5→4 domain with the cpAvd6→5 domain, and with the A subunits of two known avidin−biotin complex structures (PDB code 2AVI (light grey) and 1AVD (grey)). The N and C termini of the native avidin structures are labelled with an asterisk. Other labels and colouring are as in (b). Only the biotin of cpAvd5→4 is shown. (d) Comparison of the biotin binding modes of the cpAvd5→4 domain, cpAvd6→5 domain, 2AVI and 1AVD structures. The residues are labelled according to wt avidin. The label for residue Phe72, which is in a clearly different conformation in the cpAvd6→5 domain compared to the other shown structures, is indicated with a yellow background. The weighted difference F[o]−F[c] electron density map (cyan), calculated in the absence of biotin, is drawn with a 2.2 Å radius around the atoms of D-biotin of the final structure of the cpAvd5→4 domain. Only side-chains of amino acids are shown except for residues 37−40 of the loop between β-strands three and four.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 359, 1352-1363) copyright 2006.

Figures were selected by an automated process.