PDBsum entry 1w16

Metal binding protein

PDB id: 1w16

Contents

Protein chain

125 a.a. *

Metals

_CL ×7

_NA ×2

Waters ×53

* Residue conservation analysis

PDB id:

1w16

Name:

Metal binding protein

Title:

Rat synaptotagmin 4 c2b domain in the absence of calcium

Structure:

Synaptotagmin iv. Chain: a. Fragment: c2b domain, residues 288-425. Engineered: yes

Source:

Rattus norvegicus. Rat. Organism_taxid: 10116. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.30Å R-factor: 0.217 R-free: 0.257

Authors:

H.Dai,O.-H.Shin,M.Machius,D.R.Tomchick,T.C.Sudhof,J.Rizo

Key ref:

H.Dai et al. (2004). Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4. Nat Struct Mol Biol, 11, 844-849. PubMed id: 15311271 DOI: 10.1038/nsmb817

Date:

16-Jun-04 Release date: 13-Aug-04

Protein chain

P50232  (SYT4_RAT) -  Synaptotagmin-4 from Rattus norvegicus

Seq: 425 a.a.

Struc: 125 a.a.

DOI no: 10.1038/nsmb817

Nat Struct Mol Biol 11:844-849 (2004)

PubMed id: 15311271

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4.

H.Dai, O.H.Shin, M.Machius, D.R.Tomchick, T.C.Südhof, J.Rizo.

ABSTRACT

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.

Selected figure(s)

Figure 2.
Figure 2. Intrinsic Ca^2+-binding properties of the synaptotagmin 4 and 11 C[2] domains. (a -f) 1H-15N HSQC spectra obtained at different Ca^2+ concentrations for the rat synaptotagmin 4 C[2]A (a) and C[2]B (b) domains, the D. melanogaster synaptotagmin 4 C[2]A (c) and C[2]B (d) domains, and the rat synaptotagmin 11 C[2]A (e) and C[2]B (f) domains. The protein concentrations were 120 M and the total Ca^2+ concentrations used (mM) were: a, 0, 0.2, 1, 5, 10, 20, 40, 80; b, 0, 20; c, 0, 5, 10, 20, 40, 80; d, 0, 0.2, 1, 3, 10, 20, 40; e, 0, 0.2, 1, 3, 5, 10, 20, 40, 80; f, 0, 20. The 1H-15N HSQC spectra acquired in the absence of Ca^2+ and the highest Ca^2+ concentrations are shown with multiple red contours and multiple black contours, respectively, whereas spectra obtained at intermediate Ca^2+ concentrations are shown with single black contours.

Figure 4.
Figure 4. Crystal structure of the rat synaptotagmin 4 C[2]B domain. (a) Ribbon diagram of the rat synaptotagmin 4 C[2]B domain. Strands (cyan) are labeled 1 -8 and the single helix (orange) is labeled HA. (b) Electron density map of the Ca^2+-binding site of the rat synaptotagmin 4 C[2]B domain. Contours are drawn at 1.0 the r.m.s. level of the map. Oxygen atoms are red, nitrogen atoms are blue and carbon atoms are yellow. The Ca^2+ ion is labeled Ca, the protein Ca^2+ ligands are labeled D2, D3 and E4, and a coordinating water molecule is labeled W. (c) Backbone superposition of the structures of the rat synaptotagmin 4 C[2]B domain (orange) and the rat synaptotagmin 1 C[2]B domain8 (cyan). (d) Superposition of the Ca^2+-binding loops of the rat synaptotagmin 4 C[2]B domain (orange) and the rat synaptotagmin 1 C[2]B domain (cyan). (e) Superposition of the Ca^2+-binding loops of the rat synaptotagmin 4 C[2]B domain (orange) and the rat synaptotagmin 1 C[2]A domain (dark blue). In d and e, the Ca^2+ ions are in the same color as the protein. The figure was generated with InsightII (MSI) and MolScript35.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2004, 11, 844-849) copyright 2004.

Figures were selected by the author.