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. 2007 May;16(5):807-14.
doi: 10.1110/ps.072752407. Epub 2007 Mar 30.

Solution structure of human sorting nexin 22

Jikui Song  1 , Kate Qin Zhao, Carrie L Loushin Newman, Dmitriy A Vinarov, John L Markley
Affiliations

Solution structure of human sorting nexin 22

Jikui Song et al. Protein Sci. 2007 May.

Abstract

The sorting nexins (SNXs) constitute a large group of PX domain-containing proteins that play critical roles in protein trafficking. We report here the solution structure of human sorting nexin 22 (SNX22). Although SNX22 has <30% sequence identity with any PX domain protein of known structure, it was found to contain the alpha/beta fold and compact structural core characteristic of PX domains. Analysis of the backbone dynamics of SNX22 by NMR relaxation measurements revealed that the two walls of the ligand binding cleft undergo internal motions: on the picosecond timescale for the beta1/beta2 loop and on the micro- to millisecond timescale for the loop between the polyproline motif and helix alpha2. Regions of the SNX22 structure that differ from those of other PX domains include the loop connecting strands beta1 and beta2 and the loop connecting helices alpha1 and alpha2, which appear to be more mobile than corresponding loops in other known structures. The interaction of dibutanoyl-phosphatidylinositol-3-phosphate (dibutanoyl-PtdIns(3)P) with SNX22 was investigated by an NMR titration experiment, which identified the binding site in a basic cleft and indicated that ligand binding leads only to a local structural rearrangement as has been found with other PX domains. Because motions in the loops are damped out when dibutanoyl-PtdIns(3)P binds, entropic effects could contribute to the lower affinity of SNX22 for this ligand compared to other PX domains.

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Figures

Figure 1.
Figure 1.
Solution structure and backbone mobility of human sorting nexin 22 (SNX22). (A) Stereoview of the ensemble of 20 conformers representing the structure colored to distinguish residues in helices (red), β-strands (green), and loops (gray). (B) Ribbon representation of a representative conformer (that with the lowest energy), with individual elements of secondary structure labeled and colored as in (A) and the proline-rich sequence (V60–K66) in yellow. For clarity, the disordered eight residues from the N terminus and eight residues from the C terminus are not shown.
Figure 2.
Figure 2.
15N R2/R1 ratios and 15N-NOEs for SNX22 plotted as a function of residue number.
Figure 3.
Figure 3.
(A) Superimposed structures of: (left) SNX22 (red; this work) and Grd19p (blue; Zhou et al. 2003); (middle) SNX22 (red) and P40phox (green; Bravo et al. 2001); (right) Grd19p (blue) and P40phox (green). (B) Alignment of PX domain sequences with residues colored to indicate those that are strictly conserved (red highlighted in yellow), identical in two of the three sequences (green highlight), and similar (cyan highlight).
Figure 4.
Figure 4.
(A) Overlaid 2D [1H,15N]-HSQC spectra highlight the progressive chemical shift changes of selected residues (dashed lines). The spectra were collected for 0.2 mM [U-15N,13C]-SNX22 in the presence of various concentrations of dibutanoyl-PtdIns(3)P: (red) 0 mM, (orange) 0.1 mM, (green) 0.2 mM, (cyan) 0 4 mM, (blue) 0.8 mM, (purple) 1.6 mM. (B) Plot of Δδave for S43 and Y44 as a function of the concentration of the dibutanoyl-PtdIns(3)P. (C) Ribbon representation of the solution structure of SNX22 showing the residues (red) that display the largest chemical shift changes (Δδave > 0.05 ppm) between samples containing 0 mM and 1.6 mM dibutanoyl-PtdIns(3)P. Signal broadening prevented observation of the NMR peak from R67 (yellow). (D) Surface representation of the structure SNX22:dibutyl-PtdIns(3)P complex generated from the experimental NMR data by the program HADDOCK (Dominguez et al. 2003). The 1- and 3-phosphate groups, and the 4- and 5-OH groups of dibutanoyl-PtdIns(3)P are colored red and green, respectively.
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