Comment on "A G Protein Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid"

doi:10.1126/science.1143230

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Science 9 November 2007:
Vol. 318. no. 5852, p. 914
DOI: 10.1126/science.1143230

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Technical Comments

Comment on "A G Protein–Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid"

Christopher A. Johnston¹, Brenda R. Temple², Jin-Gui Chen⁴, Yajun Gao⁴, Etsuko N. Moriyama⁵, Alan M. Jones¹^,3^*, David P. Siderovski¹^* and Francis S. Willard¹^*

¹ Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA.
² Structural Bioinformatics Core Facility, University of North Carolina, Chapel Hill, NC 27599, USA.
³ Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA.
⁴ Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
⁵ School of Biological Sciences and Plant Science Initiative, University of Nebraska, Lincoln, NE 68588–0660, USA

Fig. 1. GCR2 is a member of the LanC protein superfamily. (A) Multiple sequence alignment of GCR2 and LanC family proteins. Secondary structures observed in the NisC crystal structure are denoted with the 14 major alpha helical regions ( ${alpha}$ 1 to ${alpha}$ 14) of NisC underlined in red, the ß strands (ß1-3) of the SH2-like "extended" domain underlined in blue, and residues involved in interhelix turns denoted by blue Ts. Conserved zinc-coordinating residues are denoted by asterisks. Proteins are denoted by their Swiss-Prot identifiers, except for GCR2 (GenBank accession NP_175700). Information for this figure was obtained from the PDB file 2G0D and Li et al. (6). Species abbreviations are ARATH (A. thaliana), BACSU (Bacillus subtilis), DROME (Drosophila melanogaster), HUMAN (Homo sapiens), LACLA (L. lactis), and STAEP (Staphylococcus epidermidis). (B) Percentage identity (orange boxes) and percentage similarity (blue boxes) from pairwise BLAST comparisons of indicated protein sequences using the BLOSUM45 matrix (4), except where the footnotes indicate identity and similarity statistics alternatively obtained from the BESTFIT algorithm (Accelrys GCG package) over the following subspans (aa = amino acids) of the indicated protein sequences: ^a, 71 aa; ^b, 123 aa; ^c, 183 aa; ^d, 66 aa; ^e, 93 aa; ^f, 117 aa; ^g, 366 aa; ^h, 74 aa; ⁱ, 113 aa; ^j, 94 aa; ^k, 355 aa; ^l, 72 aa; ^m, 110 aa; ⁿ, 65 aa; ^o, 292 aa; ^p, 55 aa; ^q, 260 aa. [View Larger Version of this Image (61K GIF file)]

Fig. 2. GCR2 has a predicted tertiary structure consistent with a LanC protein. BLASTP search against the structural database (www.rcsb.org), using GCR2 as the query, identified nisin cyclase (NisC, PDB ID: 2G0D) as the only structural homolog producing a statistically significant alignment [expect (E) value, 4 x 10^–4]. Significant sequence similarity was noted between amino acids 216 and 282 of GCR2 and amino acids 209 and 386 of NisC. A homology model of GCR2 (amino acids 216 to 282) was then generated using Insight-II (www.accelrys.com/products/insight). Shown is a superposition of the GCR2 homology model (blue) and the corresponding region of NisC (green). The N and C termini are labeled accordingly. Alpha helices observed in the NisC structure are denoted H8 to H14. Arrows indicate two segments in which NisC contains extended inserts relative to GCR2 and are the only areas of the superposition that diverge between the molecules. The proposed catalytic residues are indicated in NisC (yellow sticks) and GCR2 (red sticks). The superposition and image were generated using PyMol (DeLano Scientific, Palo Alto, CA, USA). [View Larger Version of this Image (53K GIF file)]

Fig. 3. Simulated surface plasmon resonance binding curves for a 2 nM affinity interaction between GPA1 and GCR2. Simulations of GPA1 binding to immobilized GCR2, using the rate constants published by Liu et al. (k_a = 1.77 x 104 M^–1s^–1; k_d = 3.9 x 10^–5 s^–1). Simulated injections are plotted for four different concentrations of GPA1 as reported by Liu et al. (1). Arrow indicates the injection time course and corresponding association phase. Simulated sensorgrams were generated using BIAeval 3.2 software (GE Healthcare, Uppsala, Sweden), using the 1:1 Langmuir model with maximum binding of 100 RU. (A) Simulated sensorgrams for an interaction that has no bulk buffer shift. (B) Simulated sensorgrams for an interaction occurring with a bulk buffer shift of 100 RU. [View Larger Version of this Image (25K GIF file)]