The GYF domain is a novel structural fold that is involved in lymphoid signaling through proline-rich sequences.

T cell activation through the CD2 cell surface receptor is transmitted by proline-rich sequences within its cytoplasmic tail. A membrane-proximal proline-rich tandem repeat, involved in cytokine production, is recognized by the intracellular CD2 binding protein CD2BP2. We solved the solution structure of the CD2 binding domain of CD2BP2, which we name the glycine-tyrosine-phenylalanine (GYF) domain. The GYF sequence is part of a structurally unique bulge-helix-bulge motif that constitutes the major binding site for the CD2 tail. A hydrophobic surface patch is created by motif residues that are highly conserved among a variety of proteins from diverse eukaryotic species. Thus, the architecture of the GYF domain may be widely used in protein-protein associations.     

The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

Characterization of the mechanisms controlling Greatwall activity

Here we investigate the mechanisms regulating Greatwall (Gwl), a serine/threonine kinase essential for promoting the correct timing of mitosis. We identify Gwl as a unique AGC kinase that, unlike most AGC members, appears to be devoid of a hydrophobic motif despite the presence of a functional hydrophobic pocket. Our results suggest that Gwl activation could be mediated by the binding of its hydrophobic pocket to the hydrophobic motif of another AGC kinase. Our molecular modeling and mutagenic analysis also indicate that Gwl displays a conserved tail/linker site whose phosphorylation mediates kinase activation by promoting the interaction of this phosphorylated residue with two lysines at the N terminus. This interaction could stabilize the αC-helix and maintain kinase activity. Finally, the different phosphorylation sites on Gwl are identified, and the role of each one in the regulation of Gwl kinase activity is determined. Our data suggest that only the phosphorylation of the tail/linker site, located outside the putative T loop, appears to be essential for Gwl activation. In summary, our results identify Gwl as a member of the AGC family of kinases that appears to be regulated by unique mechanisms and that differs from the other members of this family.     

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP- and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF-binding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.     

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution.

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.     

Structural basis of PxxDY motif recognition in SH3 binding

We have determined the solution structure of epidermal growth factor receptor pathway substrate 8 (Eps8) L1 Src homology 3 (SH3) domain in complex with the PPVPNPDYEPIR peptide from the CD3ε cytoplasmic tail. Our structure reveals the distinct structural features that account for the unusual specificity of the Eps8 family SH3 domains for ligands containing a PxxDY motif instead of canonical PxxP ligands. The CD3ε peptide binds Eps8L1 SH3 in a class II orientation, but neither adopts a polyproline II helical conformation nor engages the first proline-binding pocket of the SH3 ligand binding interface. Ile531 of Eps8L1 SH3, instead of Tyr or Phe residues typically found in this position in SH3 domains, renders this hydrophobic pocket smaller and nonoptimal for binding to conventional PxxP peptides. A positively charged arginine at position 512 in the n-Src loop of Eps8L1 SH3 plays a key role in PxxDY motif recognition by forming a salt bridge to D7 of the CD3ε peptide. In addition, our structural model suggests a hydrogen bond between the hydroxyl group of the aromatic ring of Y8 and the carboxyl group of E496, thus explaining the critical role of the PxxDY motif tyrosine residue in binding to Eps8 family SH3. These finding have direct implications also for understanding the atypical binding specificity of the amino-terminal SH3 of the Nck family proteins.     

The SH3 domain of a M7 interacts with its C-terminal proline-rich region

Myosins play essential roles in migration, cytokinesis, endocytosis, and adhesion. They are composed of a large N-terminal motor domain with ATPase and actin binding sites and C-terminal neck and tail regions, whose functional roles and structural context in the protein are less well characterized. The tail regions of myosins I, IV, VII, XII, and XV each contain a putative SH3 domain that may be involved in protein-protein interactions. SH3 domains are reported to bind proline-rich motifs, especially "PxxP" sequences, and such interactions serve regulatory functions. The activity of Src, PI3, and Itk kinases, for example, is regulated by intramolecular interactions between their SH3 domain and internal proline-rich sequences. Here, we use NMR spectroscopy to reveal the structure of a protein construct from Dictyostelium myosin VII (DdM7) spanning A1620-T1706, which contains its SH3 domain and adjacent proline-rich region. The SH3 domain forms the signature beta-barrel architecture found in other SH3 domains, with conserved tryptophan and tyrosine residues forming a hydrophobic pocket known to bind "PxxP" motifs. In addition, acidic residues in the RT or n-Src loops are available to interact with the basic anchoring residues that are typically found in ligands or proteins that bind SH3 domains. The DdM7 SH3 differs in the hydrophobicity of the second pocket formed by the 3(10) helix and following beta-strand, which contains polar rather than hydrophobic side chains. Most unusual, however, is that this domain binds its adjacent proline-rich region at a surface remote from the region previously identified to bind "PxxP" motifs. The interaction may affect the orientation of the tail without sacrificing the availability of the canonical "PxxP"-binding surface.     

The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation

The regulation of the activity of Abl and Src family tyrosine kinases is mediated by intramolecular interactions between the SH3, SH2, and kinase (SH1) domains. We have determined the crystal structure of an unphosphorylated form of c-Src in which the SH2 domain is not bound to the C-terminal tail. This results in an open structure where the kinase domain adopts an active conformation and the C terminus binds within a hydrophobic pocket in the C-terminal lobe. NMR binding studies support the hypothesis that an N-terminal myristate could bind in this pocket, as observed for Abl, suggesting that c-Src may also be regulated by myristate binding. In addition, the structure contains a des-methyl analog of the antileukemia drug imatinib (STI571; Gleevec). This structure reveals why the drug shows a low affinity for active kinase conformations, contributing to its excellent kinase selectivity profile.     

Crystal structure of the N-terminal SH3 domain of mouse betaPIX, p21-activated kinase-interacting exchange factor

The mouse betaPIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3(2)21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact beta-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse betaPIX-SH3 domain binding the way in which the betaPIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse betaPIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse betaPIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of betaPIX-SH3 domain resulting in its multiple biological functions.     

A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.     

Recognition of accessory protein motifs by the gamma-adaptin ear domain of GGA3

Adaptor proteins load transmembrane protein cargo into transport vesicles and serve as nexuses for the formation of large multiprotein complexes on the nascent vesicles. The gamma-adaptin ear (GAE) domains of the AP-1 adaptor protein complex and the GGA adaptor proteins recruit accessory proteins to these multiprotein complexes by binding to a hydrophobic motif. We determined the structure of the GAE domain of human GGA3 in complex with a peptide based on the DFGPLV sequence of the accessory protein Rabaptin-5 and refined it at a resolution of 2.2 A. The leucine and valine residues of the peptide are partly buried in two contiguous shallow, hydrophobic depressions. The anchoring phenylalanine is buried in a deep pocket formed by the aliphatic portions of two conserved arginine residues, along with an alanine and a proline, illustrating the unusual function of a cluster of basic residues in binding a hydrophobic motif.     

A novel RNA-binding mode of the YTH domain reveals the mechanism for recognition of determinant of selective removal by Mmi1

The YTH domain-containing protein Mmi1, together with other factors, constitutes the machinery used to selectively remove meiosis-specific mRNA during the vegetative growth of fission yeast. Mmi1 directs meiotic mRNAs to the nuclear exosome for degradation by recognizing their DSR (determinant of selective removal) motif. Here, we present the crystal structure of the Mmi1 YTH domain in the apo state and in complex with a DSR motif, demonstrating that the Mmi1 YTH domain selectively recognizes the DSR motif. Intriguingly, Mmi1 also contains a potential m⁶A (N⁶-methyladenine)-binding pocket, but its binding of the DSR motif is dependent on a long groove opposite the m⁶A pocket. The DSR-binding mode is distinct from the m⁶A RNA-binding mode utilized by other YTH domains. Furthermore, the m⁶A pocket cannot bind m⁶A RNA. Our structural and biochemical experiments uncover the mechanism of the YTH domain in binding the DSR motif and help to elucidate the function of Mmi1.     

Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway

The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-A resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.     

High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site

3-phosphoinositide dependent protein kinase-1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 A crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the "PIF-pocket", which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C-terminal PIF-pocket-interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF-pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site-directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate-dependent docking site. We discuss the possibility that an analogous phosphate-binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.     

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.     

The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms beta-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other.     

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.     

The switch helix: A putative combinatorial relay for interprotomer communication in cGMP-dependent protein kinase

For over three decades the isozymes of cGMP-dependent protein kinase (PKG) have been studied using an array of biochemical and biophysical techniques. When compared to its closest cousin, cAMP-dependent protein kinase (PKA), these studies revealed a set of identical domain types, yet containing distinct, sequence-specific features. The recently solved structure of the PKG regulatory domain showed the presence of the switch helix (SW), a novel motif that promotes the formation of a domain-swapped dimer in the asymmetric unit. This dimer is mediated by the interaction of a knob motif on the C-terminal locus of the SW, with a hydrophobic nest on the opposing protomer. This nest sits adjacent to the cGMP binding pocket of the B-site. Priming of this site by cGMP may influence the geometry of the hydrophobic nest. Moreover, this unique interaction may have wide implications for the architecture of the inactive and active forms of PKG. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).