Structural basis for the interaction between dynein light chain and the glutamate channel homolog GRINL1A ´ ´ ´ ´ ´ Marıa F Garcıa-Mayoral1, Monica Martınez-Moreno2, Juan P Albar3, Ignacio Rodrıguez-Crespo2 and Marta Bruix1 ´ ´ ´ Departamento de Espectroscopıa y Estructura Molecular, Instituto de Quımica-Fısica Rocasolano, Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain ´ ´ Departamento de Bioquımica y Biologıa Molecular I, Universidad Complutense, Madrid, Spain ´ ´ Proteomics Facility, Centro Nacional de Biotecnologıa, Consejo Superior de Investigaciones Cientıficas, Madrid, Spain Keywords dynein light chain; glutamate channel homolog; NMR; protein–protein interactions Correspondence ´ I Rodrıguez-Crespo, Departamento de ´ ´ Bioquımica y Biologıa Molecular I, Universidad Complutense, 28040 Madrid, Spain Fax: +34 913944159 Tel: +34 913944137 E-mail: nacho@bbm1.ucm.es ´ M Bruix, Departamento de Espectroscopıa y Estructura Molecular, Instituto de ´ ´ Quımica-Fısica Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain Fax: +34 915642431 Tel: +34 917459511 E-mail: mbruix@iqfr.csic.es (Received 12 January 2010, revised 11 March 2010, accepted 15 March 2010) doi:10.1111/j.1742-4658.2010.07649.x Human dynein light chain (DYNLL1) is a dimeric 89-residue protein that is known to be involved in cargo binding within the dynein multiprotein complex Over 20 protein targets, of both cellular and viral origin, have been shown to interact with DYNLL1, and some of them are transported in a retrograde manner along microtubules Using DYNLL1 as bait in a yeast two-hybrid screen with a human heart library, we identified GRINL1A (ionotropic glutamate receptor N-methyl-d-aspartate-like 1A), a homolog of the ionotropic glutamate receptor N-methyl d-aspartate, as a DYNLL1 binding partner Binding of DYNLL1 to GRINL1A was also demonstrated using GST fusion proteins and pepscan membranes Progressive deletions allowed us to narrow the DYNLL1 binding region of GRINL1A to the sequence REIGVGCDL Combining these results with NMR data, we have modelled the structure of the GRINL1A–DYNLL1 complex By analogy with known structures of DYNLL1 bound to BCL-2interacting mediator (BIM) or neuronal nitric oxide synthase (nNOS), the GRINL1A peptide also adopts an extended b-strand conformation that expands the central b-sheet within DYNLL1 Structural comparison with the nNOS–DYNLL1 complex reveals that a glycine residue of GRINL1A occupies the conserved glutamine site within the DYNLL1 binding groove Hence, our data identify a novel membrane-associated DYNLL1 binding partner and suggest that additional DYNLL1-binding partners are present near this glutamate channel homolog Structured digital abstract l MINT-7713396: DYNLL1 (uniprotkb:P63167) and GRINL1A (uniprotkb:P0CAP1) bind (MI:0407) by nuclear magnetic resonance (MI:0077) l MINT-7713280, MINT-7713382: DYNLL1, (uniprotkb:P63167) physically interacts (MI:0915) with GRINL1A (uniprotkb:P0CAP1) by two hybrid (MI:0018) l MINT-7713416, MINT-7713439: GRINL1A (uniprotkb:P0CAP1) binds (MI:0407) to DYNLL1 (uniprotkb:P63167) by peptide array (MI:0081) l MINT-7713307: GRINL1A (uniprotkb:P0CAP1) binds (MI:0407) to DYNLL1 (uniprotkb:P63167) by pull down (MI:0096) Abbreviations DTT, dithiothreitol; DYNLL, dynein light chain; GKAP, guanylate kinase-associated protein; GRINL1A, ionotropic glutamate receptor N-methyl-D-aspartate-like 1A; GSH, reduced glutathione; GST, glutathione S-transferase; NMDA, N-methyl D-aspartate; nNOS, neuronal nitric oxide synthase; PAK1, p21-activated kinase 1; PSD, post-synaptic density 2340 FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS ´ M F Garcıa-Mayoral et al DYNLL1 and GRINL1A interaction Introduction Dynein light chain, which has two isoforms termed DYNLL1 and DYNLL2 in mammals, is a small dimeric protein that was initially described as a member of the myosin V [1,2] and dynein molecular motors [3–5] Originally, DYNLL1 was thought to bind to certain protein cargos and transport them in a retrograde manner along microtubules, bound to the dynein machinery However, DYNLL1 can also be found in its soluble form and not associated with the large dynein motor or microtubules [3,6] In addition, certain viruses are known to hijack the dynein machinery during their infective cycles, and several viral proteins are known to bind to DYNLL1 directly, such as rabies virus P protein [7], African swine fever p54 protein [8] or Ebolavirus protein VP35 [9] Numerous crystal and NMR atomic structures of dynein, both in the presence and absence of peptide ligands, are now available [10–13] Sequence inspection of DYNLL1-associated proteins followed by yeast two-hybrid assay, pepscan, site-directed mutagenesis and pull-down assays have revealed that DYNLL1 associates with its target proteins essentially through the sequence motifs (R ⁄ K)STQT and (K ⁄ R)(D ⁄ E)TGIQVDR [11,14,15] In both cases, the polypeptide stretches that associate with DYNLL1 adopt an extended conformation and form an additional b-strand that extends a pre-formed b-sheet, with the glutamine residue occupying an invariant position in the DYNLL1 binding groove DYNLL1 and DYNLL2 are also known to associate on the cytosolic face of the plasma membrane, mostly at the post-synaptic density, where they seem to be involved in protein clustering in the proximity of the N-methyl d-aspartate (NMDA) receptor–postsynaptic density 95 (PSD-95) complex For instance, DYNLL2, and to a lesser extent DYNLL1, bind to a PSD-95-associated protein guanylate kinase domainassociated protein (GKAP) [16] DYNLL1 also binds to neuronal nitric oxide synthase (nNOS), another PSD-95-associated protein also present in the post-synaptic density [17,18] Likewise, at inhibitory synapses, receptors for either glycine or c-amino butyric acid co-localize with the post-synaptic scaffolding protein gephyrin, another DYNLL1-associated protein [19,20] Our proteomic studies have shown that, in rat brain, DYNLL1 can associate with several NMDA receptors, such as the NR3A-2 isoform [19] In this paper, we describe the screening of a human heart library using human DYNLL1 as bait Several independent clones of the potential ionotropic glutamate receptor-like gene N-methyl-d-aspartate-like 1A (GRINL1A) were retrieved corresponding to potential DYNLL1-associated proteins Then, using the pepscan technique, we narrowed down the DYNLL1 binding site, and showed that it is localized close to the cytosolic C-terminus of GRINL1A Further complementary approaches, including GST fusion, yeast two-hybrid assays and NMR titrations, confirmed the interaction and allowed fine mapping of the DYNLL1 binding site within GRINL1A Finally, all the experimental evidence was combined to build a structural model of DYNLL1 bound to a GRINL1A peptide Results Identification of GRINL1A as a new DYNLL1-interacting protein Human DYNLL1 was fused in-frame to the binding domain of GAL4 in the prey vector pGBT9, which was then used to transform yeast After mating with a yeast pre-transformed human heart library, positive clones were selected and analysed by automated DNA sequencing Several overlapping clones were identified that included residues 144–463 of the human GRINL1A gene (Fig 1A, accession number GI:238064959), which encodes a protein of 463 amino acids homologous to ionotropic glutamate receptors [21] In order to identify potential DYNLL1 binding sites within the GRINL1A sequence, the pepscan assay was used Overlapping dodecapeptides covering residues 156–466 of the prey protein were synthesized on A Fig Identification of GRINL1A as a DYNLL1-interacting protein and fine mapping of the binding site (A) Using a yeast two-hybrid screen, residues 144–463 of GRINL1A (C-terminal end) were shown to bind to DYNLL1 (B) Pepscan analysis of the putative DYNLL1 binding site within the GRINL1A sequence B FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS 2341 ´ M F Garcıa-Mayoral et al DYNLL1 and GRINL1A interaction a cellulose membrane, which was subsequently incubated with purified recombinant DYNLL1 The binding assay of DYNLL1 to GRINL1A revealed three potential binding regions, which are all close to the C-terminus of the protein (Fig 1B) The three stretches comprised spot C4 [residues ASASLRERIRHL(342– 353)], spot C30 [residues TREIGVGCDLLP(420–431)] and spot D7 [residues VMPSRNYTPYTR(441–452)] None of the peptides has a consensus DYNLL1-binding site such as KSTQT or KDTGIQVDR [14,15], and no glutamine residues were found in these three positive spots We considered spot C4 to be a false-positive, and discarded it on the basis that peptides enriched in His, Lys and Arg amino acids typically bind to the antibody against hexahistidine used in the development of the pepscan assay With that in mind, we fused the 100 C-terminal amino acids of GRINL1A (residues 363–463) to glutathione S-transferase (GST), and performed an in vitro binding assay to DYNLL1 (Fig 2A) Purified GST–GRINL1A(363463) was bound to a glutathione–agarose resin, and a solution of purified recombinant DYNLL1 was passed through the column Elution of GST– GRINL1A(363–463) from the column using reduced glutathione (GSH) allowed us to detect DYNLL1 associated with GRINL1A by Coomassie staining (Fig 2A, lanes B and C) or by using DYNLL1specific antibodies (data not shown) This observation demonstrates that the binding site of DYNLL1 within GRINL1A is located between residues 363 and 463 DYNLL1 did not interact with GST alone bound to the glutathione–agarose resin (data not shown) Next, we analysed this interaction in detail using a yeast two-hybrid approach, with wild-type DYNLL1 fused to the bait vector pGBT9 and various GRINL1A constructs fused to the prey vector pGAD Positive interactions were confirmed by means of the X-Gal assay using double transformants that were able to grow in the absence of Trp, Leu and His Sequential shortening narrowed the binding site of DYNLL1 within the GRINL1A polypeptide to residues 365–463 (Fig 2B) We produced several point mutations, including the glutamine embedded in the consensus DYNLL1-binding motif PSQT (Q433G), and residues contained within spots D7 and D8 (R445G, Y447G and the double mutant Q433G ⁄ R445G), and determined that these mutants still bind to DYNLL1 Finally, we shortened GRINL1A and tested the segment 423–463 for DYNLL1 binding As shown in Fig 2B, this construct did not result in a positive interaction This indicates that the binding site is located between residues 365 2342 A B C Fig Binding of DYNLL1 to a GST–GRINL1A construct and fine mapping of the binding site GST–GRINL1A(363-463) was expressed and purified in Escherichia coli, and extensively dialysed (A) Purified protein was loaded onto a GSH–agarose resin Lane A, flow-through (unbound protein) Purified recombinant DYNLL1 was allowed to bind to the immobilized GST– GRINL1A(363–463), and, after extensive washing, the proteins were eluted with GSH (lanes B and C) A control of DYNLL1 only was loaded in lane D (B) Yeast two-hybrid screen of several GRINL1A constructs performed with wild-type DYNLL1 Positive binding is represented by a ‘+’ symbol (C) Comparison between GRINL1A and a set of known peptide sequences from diverse DYNLL1-interacting targets and 423, or the positive spot C30 in the pepscan assay (residues 420–431) is indeed the binding site and residues 421 and 422 (absent in the yeast two-hybrid construct) are necessary for binding To test this hypothesis, we decided to narrow down the binding site using three partially overlapping synthetic peptides in solution that were allowed to bind FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS ´ M F Garcıa-Mayoral et al to purified 15N-labelled recombinant DYNLL1 We tested peptides with the sequences VETREIGVGCD LLPS(418–432), LLPSQTGRTREIVMP(429–443) and SRNYTPYTRVLELTM(444–458) Strong binding was observed for the first peptide only (see below) Interestingly, a sequence comparison with the nNOS stretch known to bind to DYNLL1 revealed a clear sequence homology, with acidic, basic and b-branched amino acids occupying similar positions (Fig 2C) Remarkably, in the GRINL1A sequence, a glycine residue appears at the position of the glutamine residue that is present in many DYNLL1-binding proteins NMR titration of DYNLL1 with GRINL1A peptides In order to identify specific DYNLL1-interacting sequences in GRINL1A, we performed NMR titrations with the three overlapping synthetic peptides spanning the residues 418–458 of GRINL1A We recorded 15N-HSQC spectra at increasing peptide: DYNLL1 and GRINL1A interaction protein ratios using the 15N-labelled protein and unlabelled peptides The 15N-HSQC spectrum of free DYNLL1 was used for spectral assignment and to monitor changes upon addition of the peptides The spectral assignment confirmed that, under the experimental conditions used, DYNLL1 is a symmetric dimer When DYNLL1 was titrated with peptides LLPSQTGRTREIVMP and SRNYTPYTRVLELTM, no changes in resonance chemical shifts or in the signal intensity were observed, indicating that no interaction takes place However, titration with peptide VETREIGVGCDLLPS results in large chemical shift changes for numerous resonances, providing evidence for a specific interaction (Fig 3) For simplicity, we refer to the monomers as A and B, respectively The residues participating in the interaction are: (a) I8– E15, (b) W54–H68 and (c) Y75–F86 of monomer A, and (d) N33–I38 and (e) K43–K48 of monomer B These regions are found mainly in the N-terminal loop (a) and the central b-sheet (b, c) of monomer A, and helix a2 (d, e) of monomer B Fig NMR titration of DYNLL1 with the GRINL1A VETREIGVGCDLLPS peptide Superposition of 15N-HSQC spectra of DYNLL1 recorded at various DYNLL1: GRINL1A peptide VETREIGVGCDLLPS ratios during titration The spectra correspond to free DYNLL1 (red), a : ratio of protein:peptide (cyan) and excess peptide (protein:peptide ratio of approximately : 8) (blue) The inset indicates the chosen spectral region, with selected labelled residues in the slow exchange regime upon complex formation One resonance per residue is observed for free DYNLL1 (red) and in the presence of excess peptide (blue), while two resonances appear for the under-saturated complex at the : ratio (cyan) FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS 2343 ´ M F Garcıa-Mayoral et al DYNLL1 and GRINL1A interaction The exchange regime for the residues mentioned above is slow in the NMR time scale Two resonances per residue are observed at peptide:protein ratios of 0.5 and 1, corresponding to the free and bound forms, respectively Their intensities vary according to the population fractions of the free and bound forms of DYNLL1 at the various peptide: protein ratios A unique set of resonances is observed at a molar ratio of : 1, when all the peptide is bound to the protein dimer, confirming the : stoichiometry of the functional complex under these sample conditions Model of the DYNLL1–GRINL1A VETREIGVGCDLLPS peptide complex B Haddock docking calculations produced structures for the complex that were classified into four clusters Analysis of the various clusters allowed us to select the one with the highest Haddock score as the most representative model of the DYNLL1–GRINL1A VETREIGVGCDLLPS peptide complex In this cluster, the GRINL1A peptide is aligned anti-parallel to the interacting b3 strand of one DYNLL1 monomer, as observed in all other DYNLL1 complexes reported so far [10,11,15,22] In two of the other clusters, an alternative peptide orientation was obtained, and the anti-parallel orientation was observed in the remaining cluster, but the interacting surface was shifted, which disrupted the hydrogen bond network and reduced the buried interface area Figure 4A shows the 20 lowest-energy conformers from the selected cluster modelled using the Haddock docking calculations The rmsd values for the 20 lowest-energy conformers of the best cluster are ˚ ˚ 0.71 ± 0.11 A for the backbone and 1.11 ± 0.09 A for the heavy atoms The mean total energy of the ensemble is )8146 kcalỈmol)1, being )314 kcalỈmol)1 the intermolecular contribution The corresponding values for the van der Waals’ and electrostatic terms are )887 and )8523 kcalỈmol)1 for the complex, and )56 and )259 kcalỈmol)1 for the intermolecular contributions, respectively The mean protein–peptide buried ˚ surface area is 1512 A2 Biochemical and mutational analysis of several DYNLL1 partners allowed the identification of two consensus sequences (K ⁄ R)XTQT and G(I ⁄ V)QV(D ⁄ E), which contain a conserved glutamine surrounded by hydrophobic residues, as DYNLL1-interacting sequences [14,15] This glutamine forms strong hydrogen bonds with two highly conserved DYNLL1 residues, Q35 and K36, at the beginning of helix a2 Structural analysis of our best cluster shows 2344 A C Fig Structural model of the DYNLL1–GRINL1A VETREIGVGCDLLPS peptide complex (A) Superposition of the 20 lowestenergy conformers from the selected cluster modelled using Haddock docking calculations Each DYNLL1 monomer is displayed in a different colour (blue and pink) The VETREIGVGCDLLPS peptide of GRINL1A is shown in green (B) Lowest-energy conformer of the family, showing the electrostatic surface of the protein Positively charged residues are shown in blue, negatively charged residues in red, and uncharged residues in white A stick representation is used for the peptide (C) Lowest-energy conformer of the family, using ribbon and stick representations for the protein and the peptide, respectively Selected side chains with important roles in the interactions are shown Residues participating in salt-bridge interactions are labelled In all cases, only the peptide that occupies the binding cavity in the front view of the complex is shown FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS ´ M F Garcıa-Mayoral et al that, although this glutamine is not present in the GRINL1A target, the interaction still occurs in the canonical mode (Fig 4B) The peptide lies along the grooves at each side of the DYNLL1 dimer interface, extending the central b-sheet through a hydrogen bond network that connects residues H68–F62 in the swapped b3 strand of DYNLL1 monomer A with residues R421–C427 of the peptide in an anti-parallel manner, similar to that described for other complexes (Fig 4C) Due to symmetry constraints, both peptides bind to each half-site in a parallel manner, which contributes to enhanced specificity, and interactions are similar if monomers A and B are exchanged; consequently interactions concerning only one peptide are discussed The residue structurally equivalent to the glutamine present in the consensus sequences, G426, is close to the side chain of K36 in monomer B; however, the different nature of glycine does not allow interactions with the residues capping helix a2 Stabilizing saltbridge interactions are found between the carboxylate group of E422 and the amine groups of K43 and K44 of monomer B, and the side chains of R421 and D12 of monomer A These residues point towards the interior of the peptide binding groove, facing opposite sides of the central b-sheet (Fig 4C) As mentioned previously, the construct starting at residue 423 did not bind to DYNLL1, and these results corroborate the importance of residues 421 and 422 for binding Discussion Biological significance Human GRINL1A was initially identified as an ionotropic glutamate receptor-like gene that mapped to chromosome 15q22.1 [21] Subsequent analysis of the transcription unit revealed that the gene comprises at least 28 exons, and its organization proved to be much more complex than previously anticipated [23] Sequence comparison has revealed that some transcripts of GRINL1A are significantly similar to those of both the neuromuscular junction protein yotiao and the N-termini of the NR2 and NR3 NMDA receptor subunits [23] In addition, GRINL1A is severely downregulated in patients with sporadic Alzheimer’s disease [24] It has been recently reported that GRINL1A and subunits of the NMDA receptor associate at the plasma membrane and are able to co-immunoprecipitate [25] Thus, GRINL1A might be enriched in the post-synaptic density, a specialized electron-dense structure underneath the post-synaptic plasma membrane of excitatory synapses, co-localizing with the NMDA receptors and close to proteins such as PSD- DYNLL1 and GRINL1A interaction 95, nNOS, shank and GKAP The fact that GRINL1A may associate with the NMDA receptor subunits and that its C-terminus can bind DYNLL1 tightly shows that this protein member of the dynein machinery can be targeted to the post-synaptic density through its association with at least three independent proteins (GKAP, nNOS and GRINL1A), and agrees with the laminar organization revealed by negative staining and immunogold labelling [26] A non-consensus sequence within GRINL1A is responsible for DYNLL1 binding in the canonical mode In this study, we have identified GRINL1A as a novel interacting physiological partner of DYNLL1 Using various biochemical and biophysical approaches, we have delimited the protein fragment responsible for this interaction, and found that it spans residues V418– S432 This sequence does not contain the glutamine residue conserved in canonical type DYNLL1-interacting sequences So far, structural information is only available for two DYNLL1 complexes with peptides that lack the conserved glutamine, the X-ray structure of p21-activated kinase (PAK1) [22] and the docked model of myosin Va [2], which is involved in actinmediated intracellular transport However, despite this distinctive feature compared with many DYNLL1 targets, sequence alignment of GRINL1A peptide VETREIGVGCDLLPS with the interacting portion of the nNOS peptide, which belongs to the GIQVD type of consensus sequence, reveals a high degree of similarity (Fig 2C) Our NMR data provide clear evidence for direct interaction between DYNLL1 and GRINL1A peptide VETREIGVGCDLLPS, and confirm a binding stoichiometry of : (peptide:DYNLL1 dimer) This result, and the fact that only one set of resonances is observed in the spectra for free and fully bound DYNLL1, prove that, under the experimental conditions used in this study, DYNLL1 is a symmetric dimer Moreover, we have mapped the interaction surface, which is similar to that reported for other DYNLL1 complexes that or not include the conserved glutamine The exchange regime of the resonances indicates that the interaction is strong and in the sub-micromolar range Estimated dissociation constants in the low and sub-micromolar range calculated by NMR titration or isothermal titration calorimetry (ITC) have been reported for a few DYNLL1–target peptide complexes, ranging from 0.6 to lm for Bim and swallow to lm for dynein intermediate chain, 10 lm for nNOS and 100 lm for PAK1 [22,27,28] In addition, a sub-micromolar dissociation constant of 0.9 lm has also been FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS 2345 ´ M F Garcıa-Mayoral et al DYNLL1 and GRINL1A interaction reported for the dynein–intermediate chain complex based on changes in the Trp fluorescence [29] Comparison of the DYNLL1–GRINL1A peptide complex with other DYNLL1–peptide complexes The 3D model for the DYNLL1–GRINL1A VETREIGVGCDLLPS peptide complex obtained using the Haddock docking calculations is consistent with the titration data and in agreement with the DYNLL1 complexes of known structure The peptide occupies the two identical concave channels on each side of the A B Fig Comparison of the DYNLL1–GRINL1A VETREIGVGCDLLPS peptide modelled complex with other DYNLL1 complex structures (A) Superposition of the lowest-energy conformers of the DYNLL1– GRINL1A VETREIGVGCDLLPS peptide modelled complex and the solution structure of the DYNLL1–nNOS peptide complex (B) Superposition of the lowest-energy conformers of the DYNLL1– GRINL1A VETREIGVGCDLLPS peptide modelled complex and the crystallographic structure of the DYNLL1–PAK1 peptide complex In both cases, the DYNLL1 dimer from the modelled complex is shown in pink and the DYNLL1 NMR ⁄ crystal structure is shown in blue The GRINL1A VETREIGVGCDLLPS peptide is shown in green, and the nNOS ⁄ PAK1 peptides are shown in purple Selected side chains with important roles in the interactions are shown 2346 dimer surface, and extends the b-sheet dimer interface Residues R421–C427 of the GRINL1A peptide adopt a b-strand conformation as assumed by residues K229–V235 of nNOS peptide, and the pattern of hydrogen bonds that link the peptide b-strand antiparallel to the swapped b3 strand of DYNLL1 is that expected from sequence alignments with peptides containing the GIQVD consensus motif Figure 5A shows the superposition of our model and that of the DYNLL1–nNOS complex determined by NMR spectroscopy The backbone rmsd obtained ˚ when the dimer is used for the superposition is 0.88 A, a value that indicates a high conformational similarity, and no important differences in peptide side chain conformations are detected The PAK1 crystal complex ˚ (Fig 5B) is also quite similar (rmsd value 1.41 A) In addition to the main backbone b-sheet hydrogen bonds, the DYNLL1–GRINL1A peptide docked structure shows several intermolecular salt-bridge interactions, particularly involving the N-terminus of the peptide These interactions may increase the stability and binding affinity and play important roles in binding specificity Similar electrostatic interactions to those established by E422 of the GRINL1A peptide are maintained by the aspartate residues of nNOS and Bim peptides, and mutation of this residue in the Bim peptide significantly reduces the binding affinity [15,30] Additionally, D230 of nNOS has been found to form a hydrogen bond with the hydroxyl group of T67 This interaction is also present in other related complexes Several studies have shown that the aspartate at position i)4 relative to the conserved glutamine is important for binding, and its substitution by other residues significantly decreased DYNLL1 binding [22,31] Charge–charge interactions with D12 are similarly maintained by K229 of nNOS and K5 of Bim peptides Hydrophobic interactions play a crucial role in highaffinity binding of DYNLL1 to most of its targets [11] Some important contacts are: I423 with V66, L84 and F73, V425 with F62, F73, Y75 and L84, and C427 with G63, Y75, Y77 and A82 The latter are also present in the nNOS–DYNLL1 complex for the equivalent I233 and V235 residues of nNOS Other interactions, although probably less important, are also expected to contribute to the binding affinity, for example those between T420 and the backbone atoms of H68, L429 and Y77 in strand b4, and between P431 and the aliphatic part of Q80 side chain in the loop connecting strands b4 and b5 As mentioned above, conserved Q234 in nNOS and other GIQVD- and KXTQT-containing peptides is replaced by a glycine in GRINL1A Structural analyses of known DYNLL1complexes have established the FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS ´ M F Garcıa-Mayoral et al importance of this glutamine, and a role in binding specificity has been proposed [11] As shown here, the Q ⁄ G substitution does not impair the interaction, indicating that the glutamine residue is not absolutely required for binding and is most likely involved in increasing binding affinity G426 of GRINL1A occupies the equivalent position to this glutamine, which is typically involved in hydrogen bonds with the nitrogen of K36 and the carboxylate of E35 that form an N-terminal cap for helix a2 in monomer B Glycine, with its minimal side chain, cannot form such interactions in the complex described here However, the short ˚ distance between G426 and G63 ⁄ K36 (< A) enables van der Waals’ contacts to be established similarly to those maintained by G424 (G232 in nNOS) with K36 and the Y65 ring The intrinsic flexibility of the glycine residue may facilitate subtle structural rearrangements that enhance binding, compensating for interactions lost through the Q fi G substitution In summary, this study has identified a peptide sequence within the GRINL1A protein that adds to the growing list of DYNLL1 target sequences lacking the conserved glutamine that is the usual hallmark of DYNLL1 binding sequences, yet binds to DYNLL1 at the same binding site and in similar fashion A hierarchy in the binding affinity of DLC8 targets has been proposed, with a decreasing order of affinity depending on the presence of both the conserved glutamine and the aspartate at position i-4, the presence of the conserved glutamine only, or the presence of the aspartate only [22] The GRINL1A peptide VETREIGVGCDLLPS target lacks both of these residues, and still binds to DYNLL1 with high affinity, suggesting that binding specificity is not as strong as previously thought This is in agreement with the wide variety of DYNLL1-interacting partners and its biological role as a multi-functional protein Further structural work is needed to shed light on the molecular basis leading to DYNLL1 target recognition Experimental procedures Materials [15N]-labelled (NH4)Cl was purchased from Cambridge Isotope Laboratories Inc (Andover, MA, USA) Glutathione Sepharose Fast Flow resin was obtained from Amersham Pharmacia Biotech (GE Healthcare Europe GmbH, Barcelona, Spain) l-leucine, l-tryptophan, l-histidine, l-lysine, uracil, adenine, X-b-Gal (5-bromo-4-chloro-3-indolyl-b-dgalactopyranoside) and gluthatione were purchased from Sigma-Aldrich (Barcelona, Spain) 3-aminotriazole was obtained from FLUKA (Sigma-Aldrich) The pre-trans- DYNLL1 and GRINL1A interaction formed MATCHMAKER library, the yeast nitrogen base without amino acids (SD medium) and yeast transformation system were purchased from BD Clontech (Mountain View, CA, USA) Pure synthetic peptides with the GRINL1A C-terminus sequences VETREIGVGCDLLPS (418–432), LLPSQTGRTREIVMP(429–443) and SRNYTPYTRVLELTM(444–458) were purchased from Thermo Scientific Yeast two-hybrid screen Saccharomyces cerevisiae strain Y190 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4D, gal80D, cyhr2, LYS2::GAL1UAS-HIS3TATA-HIS3,MEL1; URA3::GAL1UAS-GAL1TATA-lacz) was used for all yeast two-hybrid assays The pre-transformed MATCHMAKER library is a high-complexity cDNA library cloned into a yeast GAL4 activation domain (AD) vector and pre-transformed into Saccharomyces cerevisiae host strain Y187 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4D, met), gal80D, URA3::GAL1UAS-GAL1TATA-lacz,MEL1) All cloning was performed in DH5a Escherichia coli cells, and protein expression was performed in the protease-deficient BL21 (DE3) E coli strain DYNLL1 protein was used as bait in a yeast two-hybrid screen in order to identify new DYNLL1-interacting proteins We amplified full-length DYNLL1 cDNA by PCR, introducing EcoRI and SalI sites at the 5¢ and 3¢ ends of the cDNA This PCR product was ligated into pGBT9 plasmid (BD Clontech) in-frame with the DNA binding domain of the yeast transcription factor GAL4 This construction was used to transform the yeast strain Y190 (Mata) in order to identify positive interactions with proteins of a human heart cDNA library (MATCHMAKER pre-transformed library) The library comprised > · 106 independent clones inserted in the pACT2 plasmid in yeast strain Y187 (Mata) Yeasts were allowed to mate, and positive colonies were selected by plating on Leu) ⁄ Trp) ⁄ His) ⁄ SD plates in the presence of 10 mm 3-aminotriazole (TDO plates) Approximately 150 positive colonies were picked, and the interaction was confirmed by white ⁄ blue screening using X-b-Gal as the substrate The DNA of yeasts that displayed a positive interaction was isolated and amplified by PCR using oligonucleotides that annealed in the pACT2 plasmid The PCR fragments were subsequently sequenced, and the data were analysed using public databases b-galactosidase assay In order to confirm the interaction between DYNLL1 and various fragments of GRINL1A, we performed a colony lift filter assay [32] Colonies grown on plates without histidine (TDO plates) were re-grown on fresh plates with histidine at 30 °C for days, and then replicas of the plates were taken using with sterile Whatman filters, and yeasts FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS 2347 ´ M F Garcıa-Mayoral et al DYNLL1 and GRINL1A interaction were allowed to grow over the filter in the same conditions For the b-galactosidase assay, cells were subjected to a freeze ⁄ thaw cycle in liquid nitrogen to cause lysis Then lysates were incubated with the substrate of the b-galactosidase, X-b-Gal, at 30 °C, and the appearance of blue spots was observed after 1–6 h Recombinant expression of DYNLL1 and NMR sample preparation Cloning of DYNLL1 in pET-23, with a · His tag at the C-terminus of the protein, and expression of the recombinant protein were performed as described previously [18] Three samples of 15N-labelled DYNLL1 were prepared to final concentrations of approximately 50 lm in 90% H2O ⁄ 10% D2O aqueous solutions in 100 mm potassium phosphate buffer, mm dithiothreitol (DTT), pH 7.0 DTT was added to prevent disulfide-mediated protein aggregation Concentrated solutions of the three GRINL1A peptides were prepared by dissolving the peptides in 100 mm potassium phosphate buffer solutions containing approximately 16 mm DTT to prevent cysteines from forming intermolecular disulfide bridges The final concentrations of the peptide solutions were 4.6, 6.0 and 2.7 mm for peptides VETREIGVGCDLLPS, LLPSQTGRTREIVMP and SRNYTPYTRVLELTM, respectively Cloning and expression of GST–GRINL1A We cloned various fragments of GRINL1A into the pGEX-2T plasmid by digestion of pACT2-GRINL1A with EcoRI ⁄ XhoI, and ligation into pGEX2T digested with the same restriction endonucleases These constructions were used to transform BL21 (DE3) E coli competent cells We purified the recombinant proteins using the GSH–Sepharose resin, according to the manufacturer’s instructions All recombinant proteins were dialysed against NaCl ⁄ Pi to remove GSH, and these proteins were used directly to test the interaction with DYNLL1 in vitro Binding of recombinant DYNLL1 to peptide libraries synthesized on cellulose membranes Purification of recombinant DYNLL1 has been described previously [18] Mapping studies were performed using overlapping dodecapeptides corresponding to a fragment of GRINL1A prepared by automated spot synthesis (Abimed, Langenfeld, Germany) on an amino-derivatized cellulose membrane, with their C-termini immobilized via a polyethylene glycol spacer and their N-termini acetylated We performed the DYNLL1 binding as previously reported [14,33] The cellulose membranes were coated with 1% non-fat dried milk in TBS (50 mm Tris, pH 7.0, 137 mm NaCl, 2.7 mm KCl) for h at room temperature Incuba- 2348 tion with recombinant DYNLL1 (0.13 lm) was performed overnight at room temperature Subsequently, the membrane was incubated for h at room temperature with a commercial antibody against the hexahistidine tag present in the recombinant protein (1 : 100 000 dilution in TBS) Development of the membrane was performed by enhanced chemiluminiscence according to the manufacturer’s instructions The intensity of each spot was quantified using a UVI-tec digital image analyser (UVItec, Cambridge, UK) and the software UVIband V97 In all cases, spots corresponding to the dodecapeptides synthesized onto the same membrane were compared with each other Controls with antibody in the absence of recombinant DYNLL1 were performed in order to be able to subtract non-specific binding due to reactivity of the antibody against certain synthetic peptides NMR experiments Each 15N-labelled DYNLL1 protein sample was titrated with an unlabelled GRINL1A peptide Titrations were performed by recording series of 15N-HSQC spectra at 25 °C in a Bruker Avance 800 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a z-gradient cryoprobe for the free protein and for increasing peptide:protein ratios (0, 0.5, 1.0, 2.0, 4.0 and 8.0) The spectral assignment of DYNLL1 amide proton resonances was determined from published data obtained under similar sample conditions [11,12] Chemical shift perturbation analysis was performed using weighted average values for 15N and 1H chemical shifts according to the following equation: Ddav ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðDd1 H)2 ỵ ẵDd15 N)2 =10: Haddock modelling Docking of DYNLL1 with interacting GRINL1A peptide VETREIGVGCDLLPS was performed using the Haddock software web portal (available at http://haddock.chem.uu nl/services/HADDOCK/) using ambiguous interaction restraints derived from the NMR titration experiments The coordinates for DYNLL1 were taken from the PDB structure of DYNLL1 bound to nNOS peptide (PDB ID 1F96) The coordinates for the GRINL1A VETREIGVGCDLLPS peptide were modelled using the alignment mode within Swiss Model (http://swissmodel.expasy.org/) with the PDB structure of the nNOS chain D peptide complexed with DYNLL1 (PDB ID 1F96) The two-first residues of the target GRINL1A VETREIGVGCDLLPS peptide were excluded from this alignment and were not modelled Similar docking calculations were run with the full-length GRINL1A VETREIGVGCDLLPS peptide structure built from an anti-parallel b-sheet conformation using Pymol software tools (pymol.org) In order to simplify and speed FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS ´ M F Garcıa-Mayoral et al up the calculations, and due to the twofold symmetry axis of the complex, only one peptide was docked in the DYNLL1 dimer During the first step of rigid-body energy minimization, 1000 structures were generated, of which 200 were kept for the second semi-flexible simulated annealing step and final flexible water refinement Semi-flexible residues were automatically defined from an analysis of intermolecular contacts Active and passive residues for the protein interaction interface were selected on the basis of the chemical shift perturbation data and mean residue solvent accessibility Residue solvent accessibilities were calculated using the molmol program [34], and a 30% cut-off value was chosen to define the solvent-accessible surface Selected active residues for the protein were R60, N61, G63, Y65 and T67 from monomer A, and N33, K36, K44 and K48 from monomer B Passive residues were I8, K9, N10, D12, E15 and Q80 from monomer A All residues in the peptide T420-S432 were considered active In the case of the fulllength peptide, residues V418-S432 were considered active Half of the ambiguous interaction restraints were randomly deleted for each docking trial Final cluster analysis was performed to evaluate the structure quality of the docked complexes The patterns of intermolecular interactions for the full-length peptide and the modelled peptide were very similar, and no frame shift is induced by addition of two residues at the N-terminus Acknowledgements This work was supported by grants from the Minis´ terio de Ciencia e Innovacion BFU2009-10442, CTQ2008-00080 ⁄ BQU and CSD2006-00023 We would like to thank Fernando Roncal (CBM, Madrid) for synthesis of the pepscan cellulose membranes and Peter Rapali (Budapest, Hungary) for careful revision of the manuscript References Espindola FS, Suter DM, Partata LB, Cao T, Wolenski JS, Cheney RE, King SM & Mooseker MS (2000) The light chain composition of chicken brain myosin-Va: calmodulin, myosin-II essential light chains, and 8-kDa dynein light chain ⁄ PIN 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gene Cold Spring Harb Symp Quant Biol 50, 643–650 33 Martinez-Moreno M, Navarro-Lerida I, Roncal F, Albar JP, Alonso C, Gavilanes F & RodriguezCrespo I (2003) Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique FEBS Lett 544, 262–267 34 Koradi R, Billeter M & Wuthrich K (1996) molmol: a ă program for display and analysis of macromolecular structures J Mol Graph 14, 29–32 & 51–55 FEBS Journal 277 (2010) 2340–2350 ª 2010 The Authors Journal compilation ª 2010 FEBS ... values for the 20 lowest-energy conformers of the best cluster are ˚ ˚ 0. 71 ± 0 .11 A for the backbone and 1. 11 ± 0.09 A for the heavy atoms The mean total energy of the ensemble is ) 814 6 kcalỈmol )1, ... saltbridge interactions are found between the carboxylate group of E422 and the amine groups of K43 and K44 of monomer B, and the side chains of R4 21 and D12 of monomer A These residues point towards the. .. his3-200, ade2 -10 1, lys2-8 01, trp1-9 01, leu2-3, 11 2, gal4D, gal80D, cyhr2, LYS2::GAL1UAS-HIS3TATA-HIS3,MEL1; URA3::GAL1UAS-GAL1TATA-lacz) was used for all yeast two-hybrid assays The pre-transformed