PDBsum entry 1okq

Metal binding protein

PDB id: 1okq

Contents

Protein chain

374 a.a. *

Metals

_CA

Waters ×38

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Distinct requirements for heparin and alpha-Dystroglycan binding revealed by structure-Based mutagenesis of the laminin alpha2 lg4-Lg5 domain pair.

Authors

H.Wizemann, J.H.Garbe, M.V.Friedrich, R.Timpl, T.Sasaki, E.Hohenester.

Ref.

J Mol Biol, 2003, 332, 635-642. [DOI no: 10.1016/S0022-2836(03)00848-9]

PubMed id

12963372

Abstract

Laminin-2 (alpha2beta1gamma1) is found in basement membranes surrounding muscle and peripheral nerve cells. Several types of cellular receptors bind to the laminin G-like (LG) domains at the C terminus of the alpha2 chain, the interaction with alpha-dystroglycan (alpha-DG) being particularly important in muscle. We have used site-directed mutagenesis and in vitro binding assays to map the binding sites on the laminin alpha2 chain LG4-LG5 domain pair for alpha-DG, heparin and sulfatides. Calcium-dependent alpha-DG recognition requires the calcium ion in LG4, but not the one in LG5, as well as basic residues in both LG domains. Heparin and sulfatides also bind to basic residues in both LG domains, but there is little overlap in the binding sites for alpha-DG and heparin/sulfatides. The results should prove useful for the molecular dissection of laminin-receptor interactions in vivo.

Figure 1.
Figure 1. (a) Electrostatic surface representation of the laminin a2 LG4-LG5 structure.[14.] Positive and negative potential are represented by blue and red colouring, respectively. The Figure was made with GRASP. [31.] (b) Ca trace of the laminin a2 LG4-LG5 structure in the same orientation, with amino acid side-chains mutated in the present study shown as ball-and-stick models. Basic and acidic residues are coloured blue and red, respectively, and calcium ions are shown as pink spheres. The Figure was made with BOBSCRIPT [32.] and RASTER3D. [33.]

Figure 4.
Figure 4. Stereoview of the mutated calcium binding site in the laminin a2 LG4-LG5 D2808A/D2876A mutant. The wild-type structure is superimposed for comparison and shown in light blue. The F[obs] -F[calc] difference electron density map is shown in green at the +3s level.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 332, 635-642) copyright 2003.

Secondary reference #1

Title

Structure of the c-Terminal laminin g-Like domain pair of the laminin alpha2 chain harbouring binding sites for alpha-Dystroglycan and heparin.

Authors

D.Tisi, J.F.Talts, R.Timpl, E.Hohenester.

Ref.

EMBO J, 2000, 19, 1432-1440. [DOI no: 10.1093/emboj/19.7.1432]

PubMed id

10747011

Figure 2.
Figure 2 (A) The LG4–LG5 interface. The colour scheme is the same as in Figure 1. Residues involved in inter-domain contacts are shown as ball-and-stick models and are labelled. The most prominent contact is centred on Phe2931 (see the text). (B) Interactions between the N-terminal segment (in brown) and LG5 (in green). Dashed lines indicate hydrogen bonds. The electron density shown is a simulated annealing 2F[obs] - F[calc] omit map at 2.0 Å resolution (1.5 contouring), in which the N-terminal segment and Cys3017 in LG5 were excluded from the phasing model.

Figure 4.
Figure 4 Calcium-binding sites in 2LG4–5. (A) Calcium 1 in LG4. The calcium ligands are shown as ball-and-stick models and are labelled. Calcium–ligand bonds are shown as black sticks. A metal-bound water molecule is shown in yellow. (B) Calcium 2 in LG5. The calcium coordination is similar to calcium 1, but Asp2861 from a packing-related molecule in the crystal (in yellow) occupies the fifth coordination site (see the text).

The above figures are reproduced from the cited reference which is an Open Access publication published by Macmillan Publishers Ltd

Secondary reference #2

Title

Structural analysis and proteolytic processing of recombinant g domain of mouse laminin alpha2 chain.

Authors

J.F.Talts, K.Mann, Y.Yamada, R.Timpl.

Ref.

Febs Lett, 1998, 426, 71-76. [DOI no: 10.1016/S0014-5793(98)00312-3]

PubMed id

9598981

Figure 3.
Fig. 3. Electron microscopy after rotary shadowing of recombinant LG fragments. A: LG2, B: LG4, C: LG4–5 and D: LG1–3. The magnification bar (100 nm) is representative for all sections.

Figure 4.
Fig. 4. Immunoblotting of laminins with antisera raised against the G domain of the mouse laminin α2 chain. The antisera were against fragments LG1–3 (lanes 1, 2) and LG4–5 (lanes 3, 4). A mixture of human placenta laminin-2 and -4 (lanes 2, 3) and of mouse laminin-1 (lanes 1, 4) was used at 0.5 μg/lane. Samples were reduced and the electrophoresis was calibrated with reduced marker proteins indicated in kDa in the left margin.

The above figures are reproduced from the cited reference with permission from the Federation of European Biochemical Societies