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• W2036402203 abstract "Article15 March 2007free access Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif Ravi K Mahadev Ravi K Mahadev CR-UK Institute for Cancer Studies, School of Medicine, University of Birmingham, Birmingham, UK Search for more papers by this author Santiago M Di Pietro Santiago M Di Pietro Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author John M Olson John M Olson Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Hai Lan Piao Hai Lan Piao Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Gregory S Payne Corresponding Author Gregory S Payne Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Michael Overduin Corresponding Author Michael Overduin CR-UK Institute for Cancer Studies, School of Medicine, University of Birmingham, Birmingham, UK Search for more papers by this author Ravi K Mahadev Ravi K Mahadev CR-UK Institute for Cancer Studies, School of Medicine, University of Birmingham, Birmingham, UK Search for more papers by this author Santiago M Di Pietro Santiago M Di Pietro Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author John M Olson John M Olson Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Hai Lan Piao Hai Lan Piao Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Gregory S Payne Corresponding Author Gregory S Payne Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Search for more papers by this author Michael Overduin Corresponding Author Michael Overduin CR-UK Institute for Cancer Studies, School of Medicine, University of Birmingham, Birmingham, UK Search for more papers by this author Author Information Ravi K Mahadev1,‡, Santiago M Di Pietro2,‡, John M Olson2, Hai Lan Piao2, Gregory S Payne 2 and Michael Overduin 1 1CR-UK Institute for Cancer Studies, School of Medicine, University of Birmingham, Birmingham, UK 2Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA ‡These authors contributed equally to this work *Corresponding authors: Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. Tel.: +1 310 206 3121; Fax: +1 310 206 5272; E-mail: [email protected] CR-UK Institute for Cancer Studies, School of Medicine, University of Birmingham, Birmingham, B15 2TT, UK. Tel.: +44 121 414 3802; Fax: +44 121 414 4486; E-mail: [email protected] The EMBO Journal (2007)26:1963-1971https://doi.org/10.1038/sj.emboj.7601646 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein–ligand complex. The α+β structure of the protein reveals an SH3-like topology with a solvent-exposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis. Introduction Clathrin-mediated endocytosis is a major pathway for internalization of transmembrane receptors. At the molecular level, the process is orchestrated by a network of protein–protein and protein–membrane interactions centred on the coat protein clathrin and the multisubunit adaptor complex AP-2 (Traub, 2005). The modular nature of AP-2 facilitates its crucial functions in binding to clathrin, transmembrane cargo, phosphatidylinositol (4,5)-bisphosphate at the plasma membrane, and a host of accessory proteins involved in different stages of endocytosis (Owen et al, 2004; Traub, 2005). Other, more specialized adaptors internalize specific cargo proteins but lack the networking ability of AP-2. Recent studies have also pointed to an active role of the actin cytoskeleton in facilitating the process of endocytosis (Perrais and Merrifield, 2005; Kaksonen et al, 2006). Recruitment of transmembrane cargo to endocytic vesicles occurs through adaptor recognition of internalization signals present in cytoplasmic regions of the cargo proteins. In mammalian cells, several types of internalization signals have been extensively studied, including Yxxϕ and NPxY motifs (where ‘x’ is any residue and ‘ϕ’ a bulky hydrophobic residue) and ubiquitin (Hicke and Dunn, 2003; Owen et al, 2004; Traub, 2005). Each of these signals is recognized by modular domains within adaptors. Yxxϕ motifs are bound by a domain within the μ2 subunit of AP-2 adaptors, NPxY signals by the PTB domain in specialized Dab1/2 and ARH adaptors, and ubiquitin by monoubiquitin-binding domains in specialized adaptors like epsin. Two types of internalization signals have been defined in yeast: the ubiquitin-based signal recognized by monoubiquitin-binding domains (Hicke and Dunn, 2003) and the NPFxD signal, which is recognized by the Sla1p homology domain 1 (SHD1) found in Sla1p (Tan et al, 1996; Howard et al, 2002; Piao et al, 2007). Sla1p is a specialized adaptor protein which is required for the normal structure and organization of the cortical actin patches that are sites of endocytosis (Holtzman et al, 1993; Ayscough et al, 1997, 1999; Gourlay et al, 2003; Kaksonen et al, 2005). It localizes with other endocytic proteins like Sla2p, Pan1p and End3p at actin patches and interacts with a variety of proteins involved in endocytosis and modulation of actin cytoskeleton dynamics (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al, 2006). Sla1p consists of three N-terminal SH3 domains followed by SHD1 and SHD2 (unrelated to SHD1), as well as multiple C-terminal LxxQxTG repeats, which are phosphorylated by the Prk1p kinase, thus modulating Sla1p interactions (Ayscough et al, 1999; Zeng et al, 2001). SHD1 is a protein interaction domain in Sla1p that specifically recognizes NPFxD-type internalization signals found in the cytoplasmic domains of transmembrane proteins like the a-factor receptor Ste3p, the Golgi-resident furin-like protease Kex2p and the cell wall stress sensor Wsc1p (Tan et al, 1996; Howard et al, 2002; Piao et al, 2007). This interaction leads to endocytosis in an SHD1- and NPFxD-dependent manner. All four amino-acid residues defining the NPFxD signal have been shown to be important for efficient endocytosis (Tan et al, 1996; Howard et al, 2002). To better understand the molecular recognition of NPFxD by SHD1, the three-dimensional (3D) structure of Sla1p SHD1 in the presence of an NPFSD ligand sequence has been determined. Results from NMR and structure-based mutagenesis map specificity determinants for the SHD1:NPFxD interaction, allowing a model for the SHD1–NPFSD complex to be derived. The model is supported by inhibitory effects of mutating key interaction residues on ligand binding in vitro and receptor internalization in vivo. Results and discussion Solution structure of SHD1 The structure of SHD1 from Sla1p was determined in the presence of Kex2p peptide ligand using 13C, 15N-resolved NMR methods (Figure 1A and B). The fold consists of a highly twisted β-sheet followed by a C-terminal helical motif. The four antiparallel β-strands form an open barrel capped by a long α-helix bordered by two short 310 helices. The ensemble of 20 structures has a root mean square deviation (RMSD) of 0.34±0.06 Å for backbone atoms and 0.77±0.06 Å for all heavy atoms, calculated from the mean structure. Structural statistics data for SHD1 are shown in Table I. A solvent-accessible hydrophobic pocket is evident on the open face of the barrel opposite the helical cluster, and consists of residues Phe507, Val509 in β2, Lys525 in β3, Val529, and β4 residues Ile531 and Val533. The hydrophobic core of the protein is composed of Trp500 in β1, Ala511 in β2, Leu523 in β3, Ala535 in the first 310 helix, Leu538, α-helical residues Leu543 and Val546, and Leu554 in the second 310 helix. The residues in the pocket and core are highly conserved (Figure 1C), indicating a common fold and function of SHD1 domains, which are found in a variety of fungi and several bacteria. Figure 1.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. Download figure Download PowerPoint Table 1. NMR restraints and structural statistics for SHD1 Distance restraints Intraresidue (i−j=0) 222 Sequential (∣i–j∣=1) 264 Medium range (2⩽∣i−j∣⩽4) 169 Long range (∣i−j∣⩾5) 274 Hydrogen bonds 52 Total 981 Dihedral angle restraints from TALOS Phi (ϕ), Psi (ψ) 39,39 RMSD of atomic coordinates (Å) Backbone heavy atoms 0.34±0.06 All heavy atoms 0.77±0.06 RMSD from experimental restraints Distances (Å) 0.041±0.002 Dihedral angles (deg) 0.453±0.108 Violations Distances >0.5 Å and dihedral angles >5° Nil Ramachandran plot analysis, (% residues) Most favorable region 88.9 Additional allowed regions 10.1 Generously allowed regions 0.5 Disallowed regions 0.6 Model of the SHD1–NPFxD complex The interactions of the Kex2p and Ste3p peptide ligands were evident from extensive chemical shift perturbations (CSP) seen in the 15N HSQC spectra of SHD1 (Figure 2A). The Kex2p peptide, NENPFSDPIK, bound to SHD1 in the intermediate exchange regime whereas the Ste3p peptide, SRNPFSTDSE, bound more weakly, exhibiting fast exchange on the NMR timescale. Compared to the Ste3p peptide, the Kex2p peptide induced unique and more extensive perturbations in a SHD1 surface patch involving Lys525, Ala526, Asn527, Val529 and Ile531 (Figure 2B), implicating these residues as key specificity determinants for the NPFxD motif from Kex2p. Figure 2.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). Download figure Download PowerPoint The Kex2p NPFxD motif was selected for characterization of the SHD1 complex, as it bound to SHD1 with a slower apparent off-rate compared to the Ste3p NPFxxD motif. Intermolecular nuclear Overhauser effect (NOE) contacts were observed involving Val509, Val529, Ile531, Val533 and Leu538 in the 15N/13C-edited NOE and filtered NOE spectra (Figure 2C). Based on their assignments in the free Kex2p peptide, 17 unambiguous intermolecular NOEs were attributed to Kex2p residues Pro5 (six NOEs) and Phe6 (11) and SHD1 residues I531 (six NOEs), V509 (6), V529 (3), V533 (1) and L538 (1). Assignments of the remaining peptide residues in their bound states were complicated by conformational exchange and line broadening. Consequently, NOEs to the Asn4 and Asp8 ligand residues could not be unambiguously assigned, whereas other peptide residues did not show any detectable NOEs. NMR experiments with a mutant ‘NPFSA’ Kex2p peptide in which Ala was substituted for the conserved Asp suggested an Asp interaction element in the H524-A532 sequence (Figure 2A, see below), and mutagenesis of SHD1 implicated K525 in recognition of the Asp in NPFSD (Figure 3B, see below). To calculate the model of the SHD1–NPFxD complex, therefore a distance restraint of 3 Å between the ends of the K525 (SHD1) and Asp8 (NPFxD) side chains was also included. Figure 3.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. Download figure Download PowerPoint A model for the SHD1–NPFxD complex was calculated with the programme HADDOCK (Dominguez et al, 2003) using restraints based on 17 intermolecular NOEs as well as the CSPs in the 15N HSQC experiment. The ensemble of the 20 energetically most favourable structures yielded RMSDs of 0.54±0.24 and 0.87±0.31 Å for backbone and heavy atoms, respectively, spanning SHD1 Ser497–Lys558 and Kex2p Asn4–Asp8 sequences. The model of the complex is shown in Figure 2D, and depicts the Pro5 and Phe6 residues of the ligand in the centre of the hydrophobic binding site on the SHD1 surface. The Pro5 residue was positioned close to K525, Val529 and Ile531 of SHD1, whereas Phe6 packed against F507, V509, K525, I531 and V533. The Asp8 residue was oriented near K525 in SHD1 and across from Asn4, whereas the remainder of the peptide, being unconstrained, was disordered. Role of binding site residues in complex formation The contribution of SHD1 residues in and around the hydrophobic binding pocket to NPFSD recognition was investigated using surface plasmon resonance (SPR). Binding of wild-type and mutant SHD1 proteins to an NPFSD peptide was measured. SHD1 binding to NPFSD was concentration-dependent and saturable (Figure 3A), with a Kd of approximately 5 μM (Figure 3B). As expected, binding to an endocytically inactive NPASD mutant peptide was negligible (Figure 3A). Mutations F507A and I531E completely abolished the interaction whereas mutations K525A and V529E had strong effects (Figure 3A and B), emphasizing the crucial role of these residues in the binding pocket. In contrast, K508A had a weak effect and D510A a negligible effect on binding. Similar effects of mutations on binding were observed in yeast two-hybrid and GST-fusion affinity interaction assays (Figure 3C and D). These mutant SHD1 proteins were folded as assessed by circular dichroism spectroscopy, whereas mutations involving residue V509, V533 or L538 yielded insoluble or poorly folded domains following cleavage from GST. Specificity determinants for Asp in signal binding Previous work demonstrated that mutation of the Asp in NPFSD reduced but did not eliminate endocytosis mediated by the Kex2p signal, suggesting an important but not essential role for this residue in binding SHD1 (Tan et al, 1996). Additionally, weaker binding of SHD1 to the Ste3p NPFSTD peptide compared to the Kex2p peptide detected by NMR raised the possibility that spacing between the ligand Phe and Asp residues in the signal plays a role in binding efficiency. To further address the role of the Asp residue and its spacing from the NPF motif in binding, SPR was used to measure binding of NPFSA, NPFD, NPFSD, NPFSGD and NPFSGGD peptides to SHD1. As shown in Figure 3B, substitution of Ala for Asp in the Kex2p ligand, or any change in the Asp position reduced affinity by a factor of 5. Thus, both the presence and spacing of the Asp play significant although not essential roles in NPFxD signal recognition by SHD1. To identify determinants of Asp recognition, SHD1's site of interaction with the NPFSA mutant peptide was mapped by NMR. CSPs observed in the 15N HSQC spectra due to NPFSA addition were compared to those caused by the Kex2p and Ste3p peptides. Interaction of the mutant peptide resembled the binding mode of the weaker Ste3p ligand, inducing smaller perturbations in the H524-A532 region involved in wild-type Kex2p peptide binding (Figure 2A and B). This result suggested that this region harbours a specificity element for the Asp residue in the NPFxD motif. An attractive candidate is K525, which undergoes CSPs upon NPFSD binding and is important for signal binding. This interaction was further explored by measuring binding of NPFSD, NPFSGD and NPFSA peptides to the K525A mutant of SHD1. As shown in Figure 3B, the SHD1 mutant did not discriminate between the peptides, exhibiting 30-fold reductions in affinity compared to the affinity of wild-type SHD1 for NPFSD. These data strongly implicate K525 in recognition of the Asp in NPFSD, for example, through a stabilizing electrostatic interaction. However, the affinity of the K525A SHD1 mutant for NPFSD is significantly lower than that of wild-type SHD1 for the reciprocally compromised NPFSA peptide, suggesting that K525 contributes to NPFSD binding through additional interactions. SHD1 binding site residues are critical for receptor endocytosis To test the role of the hydrophobic binding pocket of SHD1 in NPFxD-dependent endocytosis in vivo, SHD1 mutations were introduced into the endogenous SLA1. Each mutant was expressed at normal levels as assessed by immunoblotting (Figure 4A). Mutant effects on endocytosis were assessed using strains expressing a chimeric plasma membrane receptor in which the NPFSD signal from Kex2p replaced the cytoplasmic C-terminal domain of the α-factor mating pheromone receptor, Ste2p (Howard et al, 2002). This receptor (Ste2pΔ318-NPFSD) exhibits normal pheromone binding but lacks the native ubiquitin-dependent targeting signals in the Ste2p cytoplasmic tail, thereby conferring complete dependence on the appended NPFSD signal for pheromone endocytosis. The internalization of Ste2pΔ318-NPFSD in wild-type and SHD1 mutant strains was monitored by measuring uptake of radiolabeled α-factor. As shown in Figure 4B, the Sla1p I531E mutation eliminated α-factor internalization whereas D510A had no effect compared to wild type. This result is consistent with the in vitro binding and NMR data that identify I531 as a critical residue for the interaction and position D510 close to, but outside, the binding pocket. Uptake of α-factor was completely abolished in K525A mutant cells and nearly absent in V529E mutant cells (Figure 4C). Considering the effects of these mutants on the affinity of SHD1 for NPFSD in vitro, the appearance of limited endocytosis in the V529E mutant (Kd 109 μM; Figure 3B) suggests that an affinity of about 100 μM is the minimum required for an effective interaction with the Ste2pΔ318-NPFSD receptor in vivo. Also in agreement with in vitro binding results, F507A mutant cells were completely defective in endocytosis and K508A mutant cells were only marginally defective (Figure 4D). Taken together, our results indicate striking parallels between effects of structure-based mutations on endocytosis in vivo and signal binding in vitro, providing strong support for the physiological significance of the NMR-derived structure. Figure 4.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. Download figure Download PowerPoint SHD1 binding site residues are important for endocytosis-dependent function of a cell wall stress sensor It has been recently reported that the major cell wall stress sensor Wsc1p relies on an NPFxD signal and SHD1 for constitutive endocytosis (Piao et al, 2007). NPFxD-dependent internalization and endocytic recycling to the plasma membrane are necessary to maintain polarized distribution of Wsc1p to sites of new cell surface growth. Mutation of the Wsc1p NPFxD signal or deletion of SHD1 from Sla1p prevents Wsc1p endocytosis and leads to uniform accumulation along the cell surface. Depolarization of Wsc1p due to defects in endocytosis compromises the ability of the sensor to respond to cell wall stress imposed by the cell wall synthesis inhibitor caspofungin (Piao et al, 2007). Thus, growth on medium containing caspofungin provides a facile and sensitive measure of NPFxD-mediated internalization of Wsc1p. Accordingly, to monitor the effects of the SHD1 binding site mutations on internalization of a native plasma membrane protein, SHD1 mutants were tested for growth in the presence of caspofungin (Figure 5A). Mutations in I531, K525, V529 and F507 caused increased sensitivity, whereas mutations in K508 and D510 did not. Figure 5.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). Download figure Download PowerPoint Wsc1p localization was directly examined in a subset of mutant strains expressing a functional Wsc1p-GFP fusion. In parallel to the caspofungin sensitivity results, mutation of I531 and K525, but not D510, caused cell surface depolarization of Wsc1p-GFP at 24°C, indicative of defective endocytosis (Figure 5B). Effects of mutations on Wsc1p-GFP internalization were more clearly evident after cells were shifted to 37°C, a treatment that stimulates sorting of internalized Wsc1p-GFP to the vacuole in wild-type cells (Piao et al, 2007). In contrast to wild-type and D510A cells, where Wsc1p-GFP was internalized and delivered to the vacuole, the protein remained uniformly distributed at the surface of I531E and K525A cells (Figure 5B). These results offer further support for the SHD1 structural model and indicate that endocytosis mediated by the NPFxD binding site plays an important physiological role in the yeast cell wall stress response pathway. SHD1 is related to domains based on the SH3 type fold Comparison of the SHD1 structure to known protein structures using DALI (Holm and Sander, 1993) identifies close structural matches to protein domains having the SH3 barrel fold, despite low sequence similarity. The closest structural neighbour is the Sm-like domain in the RNA-binding translational regulator protein Hfq (Schumacher et al, 2002) (Figure 6A). The most similar SH3 domain that binds to polyproline type II helix (PPII) motifs is the one from p40phox (Massenet et al, 2005) (Figure 6B). When the residues involved in ligand binding are compared (Figures 6C–E), the unique binding site in SHD1 is evident. In contrast to the binding site formed by residues lining β2 and β4 of SHD1, the Hfq RNA-binding site is formed by residues in the N-terminal α-helix, the loop connecting β2 and β3 and residues in the loop following β4 (close to Ala535 and Asp536 in SHD1). However, some residues lining β1 and β4 form part of the oligomerization interface (Figure 6C). Comparison to p40phox SH3 domain reveals that two residues, Pro 220 and Phe 223, occur at positions adjacent to Val533 (in β4) and Leu538, respectively, in SHD1. However, the p40phox SH3 PPII binding site also comprises crucial residues in the RT loop (Figure 6E), which is missing entirely from SHD1. Thus, SHD1 is a novel functional domain based on the SH3-like polypeptide fold that is fundamentally distinguished by its unique binding pocket, the inclusion of C-terminal helices and the absence of an RT loop. Figure 6.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 p40phox (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 p40phox-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). Download figure Download PowerPoint Sla1p as an endocytic adaptor Sla1p is required for the normal functioning of the actin cytoskeleton during endocytosis (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al, 2006). Participation in cargo binding by SHD1 recognition of NPFxD motifs makes Sla1p the only adaptor known to link endocytic cargo and actin dynamics in either yeast or mammals. The lack of obvious SHD1 motifs in higher eukaryotes suggests that its role may have been taken on by other modules, such as NPF-binding EH domains (de Beer et al, 2000) or FxNPxY-binding PTB domains (Stolt et al, 2003). Although mammalian homologues of Sla1p are not obvious by sequence comparisons, the Sla1p/Pan1p/End3p complex (which contains three SH3, SHD1, SHD2 and five EH domains) in yeast is considered functionally equivalent to the Intersectin/Eps15 complex (five SH3 and five EH domains) in mammals (Howard et al, 2002; Kaksonen et al, 2006). However, neither Intersectin nor Eps15 is known to bind directly to internalization motifs in endocytic cargo proteins. The SH3-like domain structure of SHD1 furthers the similarity of the Sla1p and Intersectin complexes in terms of" @default.
• W2036402203 created "2016-06-24" @default.
• W2036402203 creator A5025099665 @default.
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• W2036402203 date "2007-03-15" @default.
• W2036402203 modified "2023-09-27" @default.
• W2036402203 title "Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif" @default.
• W2036402203 cites W1520359022 @default.
• W2036402203 cites W1589295940 @default.
• W2036402203 cites W1973782344 @default.
• W2036402203 cites W1975459052 @default.
• W2036402203 cites W1982750125 @default.
• W2036402203 cites W1985133799 @default.
• W2036402203 cites W1988651629 @default.
• W2036402203 cites W1995017064 @default.
• W2036402203 cites W2002195659 @default.
• W2036402203 cites W2010581638 @default.
• W2036402203 cites W2012107438 @default.
• W2036402203 cites W2022058405 @default.
• W2036402203 cites W2042395036 @default.
• W2036402203 cites W2054522434 @default.
• W2036402203 cites W2058227793 @default.
• W2036402203 cites W2096447888 @default.
• W2036402203 cites W2101441813 @default.
• W2036402203 cites W2106252068 @default.
• W2036402203 cites W2106882534 @default.
• W2036402203 cites W2133987610 @default.
• W2036402203 cites W2134654436 @default.
• W2036402203 cites W2136175548 @default.
• W2036402203 cites W2137031771 @default.
• W2036402203 cites W2137745009 @default.
• W2036402203 cites W2141834983 @default.
• W2036402203 cites W2143935409 @default.
• W2036402203 cites W2144535282 @default.
• W2036402203 cites W2149739727 @default.
• W2036402203 cites W2153983456 @default.
• W2036402203 cites W2158714788 @default.
• W2036402203 cites W2163891818 @default.
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