PDBsum entry 3evv

Signaling protein

PDB id: 3evv

Contents

Protein chain

386 a.a.

Metals

_CA ×4

Waters ×54

PDB id:

3evv

Name:

Signaling protein

Title:

Crystal structure of calcium bound dimeric gcamp2 (#2)

Structure:

Myosin light chain kinase, green fluorescent protein, calmodulin chimera. Chain: a. Engineered: yes

Source:

Aequorea victoria, homo sapiens. Jellyfish, human. Organism_taxid: 6100, 9606. Gene: gfp, calm1, calm, cam, cam1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.60Å R-factor: 0.221 R-free: 0.270

Authors:

Q.Wang,B.Shui,M.I.Kotlikoff,H.Sondermann

Key ref:

Q.Wang et al. (2008). Structural basis for calcium sensing by GCaMP2. Structure, 16, 1817-1827. PubMed id: 19081058 DOI: 10.1016/j.str.2008.10.008

Date:

13-Oct-08 Release date: 09-Dec-08

Protein chain

P0DP23  (CALM1_HUMAN) -  Calmodulin-1 from Homo sapiens

Seq: 149 a.a.

Struc: 386 a.a.*

Protein chain

P11799  (MYLK_CHICK) -  Myosin light chain kinase, smooth muscle from Gallus gallus

Seq: 1906 a.a.

Struc: 386 a.a.*

Protein chain

P42212  (GFP_AEQVI) -  Green fluorescent protein from Aequorea victoria

Seq: 238 a.a.

Struc: 386 a.a.*

* PDB and UniProt seqs differ at 249 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.11.18 - [myosin light-chain] kinase.
• Reaction:
  1. L-seryl-[myosin light chain] + ATP = O-phospho-L-seryl-[myosin light chain] + ADP + H⁺
  2. L-threonyl-[myosin light chain] + ATP = O-phospho-L-threonyl-[myosin light chain] + ADP + H⁺
• Cofactor: Ca(2+)

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

reference

DOI no: 10.1016/j.str.2008.10.008

Structure 16:1817-1827 (2008)

PubMed id: 19081058

Structural basis for calcium sensing by GCaMP2.

Q.Wang, B.Shui, M.I.Kotlikoff, H.Sondermann.

ABSTRACT

Genetically encoded Ca(2+) indicators are important tools that enable the measurement of Ca(2+) dynamics in a physiologically relevant context. GCaMP2, one of the most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca(2+) fluxes in vivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveal a sophisticated molecular mechanism for Ca(2+) sensing. In the bright state, CaM stabilizes the fluorophore in an ionized state similar to that observed in EGFP. Mutational analysis confirmed critical interactions between the fluorophore and elements of the fused peptides. Solution scattering studies indicate that the Ca(2+)-free form of GCaMP2 is a compact, predocked state, suggesting a molecular basis for the relatively rapid signaling kinetics reported for this indicator. These studies provide a structural basis for the rational design of improved Ca(2+)-sensitive probes.

Selected figure(s)

Figure 1.
Figure 1. Crystal Structures of GCaMP2•Ca^2+ and cpEGFP
(A) Domain organization of GCaMP2 and truncated derivatives. A schematic presentation of the GCaMP2 fusion protein is shown. The color scheme introduced here is maintained throughout the article. Residue numbering for circularly permutated EGFP (cpEGFP) and GCaMP2ΔRSET follows the sequence of GCaMP2.
(B) Crystal structure of the isolated cpEGFP moiety. The C-terminal fragment of C-EGFP is colored in light green, and the N-terminal fragment is colored in dark green. Two orthogonal views are shown.
(C) Crystal structure of monomeric GCaMP2ΔRSET in its Ca^2+-bound state. Crystals were grown in the presence of 1 mM Ca^2+. Two orthogonal views are shown. The M13 helix is shown in blue, and the calmodulin (CaM) domain is shown in red. The cpEFGP is colored as described in (B).
(D) Comparison of crystal structures of GCaMP2, cpEGFP, and GFP-S65T. Distance difference matrices based on Cα positions were used to compare the conformation of cpEGFP in isolation (bottom-right triangle) and as part of GCaMP2 (top-left triangle) with the structure of GFP-S65T (PDB code 1EMA; see Supplemental Experimental Procedures). Difference matrices were regularized using a Z-score analysis and color-coded accordingly. Each entry in the matrix depicts the difference in distance between corresponding Cα atoms in the two structures. Distances that show little change are blue. Red entries represent distances that are significantly different in the two structures.

Figure 3.
Figure 3. Intramolecular Interfaces in Monomeric GCaMP2•Ca^2+
(A) Interfaces among the cpEGFP, M13, and CaM modules in the structure of monomeric GCaMP2ΔRSET•Ca^2+. Residues of the M13-cpEGFP module interacting with CaM are colored red. Interfacial residues on CaM are colored in green and blue for contacts with cpEGFP and the M13 helix, respectively. A top view, rotated 90° around the horizontal axis with respect to the view shown above, is shown as a cutaway rendition of the surface (bottom-left). The fluorophores of cpEGFP and Arg-377 of CaM are shown in stick presentation. Surface presentation of the isolated CaM domain and M13-cpEGFP unit were rotated by +90° and −90°, respectively, with respect to the view of the assembled structure (top-left).
(B) Electrostatic potential of the M13-cpEGFP module and CaM mapped onto its molecular surface. Views are identical to (A). Red represents negative and blue represents positive potential (−5 to +5 k[B]T).
(C) Schematic diagram of the fluorophore environment and the hydrogen bond network between cpEGFP and CaM. The numbering scheme for GCaMP2 was used. Corresponding residue numbers in GFP are shown in brackets. Carbon atoms of residues in cpEGFP, CaM, and linker segments are shown in green, dark red, and gray, respectively. Hydrogen bonds shown in the figure are between 2.7 and 3.3 Å (not drawn to scale).
(D) Close-up views of the interfacial regions in GCaMP2ΔRSET•Ca^2+. Water-mediated interaction between the fluorophore and Arg-377 of the CaM domain (top) and cpEGFP:CaM interfacial residues (bottom) are shown.

The above figures are reprinted from an Open Access publication published by Cell Press: Structure (2008, 16, 1817-1827) copyright 2008.

Figures were selected by the author.