Ligand binding to SHD1. (A) The top panel shows residue-wise CSP in SHD1 upon addition of saturating amounts of Kex2p, Ste3p and NPFSA peptides, calculated as √{(δH)²+(δN/5)²)}. The average CSPs for Kex2p () and NPFSA (- - -) binding are marked. The middle and the bottom panels show the difference in CSPs of the NPFSA peptide from those of the Kex2p and Ste3p peptides, respectively. The secondary structures along the sequence are shown on the top. (B) Surface map of perturbed residues in SHD1 upon Kex2p and Ste3p binding. Residues perturbed upon Kex2p binding are in red, those perturbed upon Ste3p binding are in yellow and residues perturbed in response to both the peptides are in orange. (C) Strips from 3D ¹⁵N/¹³C-edited NOESY and 2D ¹³C-edited, ¹⁵N/¹³C-filtered NOESY to show intermolecular NOEs for the Val509 methyl side-chain residue. (D) Model for the SHD1–NPFSD complex. SHD1 is shown in surface representation. The core NPFSD residues are shown in stick representation. Residues in SHD1 that show intermolecular NOEs and are known to be important based on mutagenesis studies are shown in deep blue. The Pro5, Phe6 and Asp8 residues are experimentally restrained and are shown in dark magenta, whereas residues Asn4 and Ser7, which are only restrained by HADDOCK, are shown in light magenta. Panels B and D were generated using PYMOL (DeLano, 2002).

NPFSD-mediated endocytosis requires SHD1 signal binding pocket residues. (A) Total yeast cell extracts from GPY1805, a sla1Δ strain (GPY2448) (Howard et al, 2002) and the Sla1p SHD1 mutant strains were analyzed by SDS–PAGE and immunoblotting with Sla1p antibodies. (B–D) Uptake of prebound, radiolabeled α-factor was measured in GPY1805 (Sla1p wild-type strain, WT) and the indicated Sla1p mutant strains expressing Ste2pΔ318-NPFSD at 30°C. Error bars represent the s.d. in two separate samples.

Representative structural neighbours of SHD1. (A, B) Sm-like domain from translational regulator Hfq (PDB ID 1KQ1) with a Z-score of 4.3 and SH3 domain from p40^phox (PDB ID 1W6X) with a Z-score of 2.1. Cα-based superposition gives an RMSD of 2.7 Å for Sm-like domain over 45 residues (with 13% sequence identity) and 2.8 Å for the SH3 domain over 40 residues (5% sequence identity). SHD1 is shown in red, the Sm-like domain in blue and the SH3 domain in green. (C–E) Surface representations Hfq, SHD1 and p40^phox-SH3 to highlight the difference in binding pockets in the three proteins. The residues, on the similar surfaces, that form the binding site are highlighted in blue, red and green, respectively. In the case of Hfq, residues involved in oligomerization are shown in yellow. All figures were generated using PYMOL (DeLano, 2002).

Solution structure of SHD1 and sequence alignment. (A) Stereoview of the NMR ensemble of 20 best structures for SHD1. Residues Arg498–Val501 in β1, Lys508–Ala517 in β2, Lys520–Lys525 in β3, Lys530–Thr550 from β4 to α1 and Leu554–Phe557 in 3₁₀2 were used for the superposition. (B) Ribbon representation of SHD1. The secondary structure elements and termini are labelled, with β-strands coloured magenta; helices, blue; and loops and termini, gray. Panels A and B were generated using MOLMOL (Koradi et al, 1996). (C) Sequence alignment of representative fungal and bacterial SHD1 sequences. Fully conserved residues are highlighted in green, partially conserved residues in yellow and substitutions with similar residues in cyan. Conserved residues in the hydrophobic core and the ligand-binding site are marked with (^*) and (▴), respectively, at the bottom of the alignment. SHD1-related sequences were identified using PSI-BLAST (Altschul et al, 1997). The alignment was created using CLUSTALW (Thompson et al, 1994) and BOXSHADE tools in the SDSC Workbench (Subramaniam, 1998). Sequences used are Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, Candida albicans and Rhodopirellula baltica. The last, [R_bal] bacterial sequence contains the least well-conserved SHD1, and the host protein lacks the SH3 domains usually found in Sla1p homologues, suggesting functional divergence. Close homologues of SHD1 are not evident in higher eukaryotes.

Mutagenesis studies on binding site residues. (A) SHD1 hydrophobic pocket residues are important for NPFSD binding. (Upper panel) Binding of SHD1 to an NPFSD peptide (▪) or an NPASD peptide (Δ) measured by SPR. (Lower panel) Binding of SHD1–K525A to an NPFSD peptide (▪) or an NPASD peptide (Δ) and SHD1–I531E to an NPFSD peptide (•). (B) The affinities of NPFSD and variants binding to wild-type and mutant SHD1, as measured by SPR, are listed. (C) NPFSD interaction with wild-type and mutant SHD1 assayed by yeast two-hybrid assays. Wild-type SHD1 and mutant versions were fused to the Gal4 activation domain (GAD) and tested for interaction with three repeats of the NPFSD signal linked to the Gal4 DNA-binding domain (GBD). Serial dilutions of cells were grown at 30°C on synthetic media containing (+His) or lacking (−His) histidine. (D) NPFSD binding to wild-type and mutant SHD1 assayed by GST fusion protein affinity. Purified wild-type SHD1 and the indicated mutants were tested for interactions with GST fused to triple repeats of active (NPFSD) or inactive (NPASD) forms of the Kex2p-derived signal. Eluted proteins were subjected to SDS–PAGE and visualized by Coomassie blue staining.

SHD1 binding pocket residues are required for Wsc1p-mediated cell wall stress response and Wsc1p endocytosis. (A) Wild-type (WT; GPY1805), sla1Δ (GPY2448) and SHD1 mutant cells were grown in YPD media and five-fold serial dilutions were spotted onto YPD or YPD containing 350 ng/ml caspofungin. (B) Wild-type (WT; GPY3527) and SHD1 mutants were grown to early log phase and then treated with or without heat shock at 37°C for 35 min. Cells were visualized by epifluorescence (left panel in each pair) and differential interference contrast (DIC) microscopy (right panel in each pair).