Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

The Shank/proline-rich synapse-associated protein family of multidomain proteins is known to play an important role in the organization of synaptic multiprotein complexes. For instance, the Shank PDZ domain binds to the C termini of guanylate kinase-associated proteins, which in turn interact with the guanylate kinase domain of postsynaptic density-95 scaffolding proteins. Here we describe the crystal structures of Shank1 PDZ in its peptide free form and in complex with the C-terminal hexapeptide (EAQTRL) of guanylate kinaseassociated protein (GKAP1a) determined at 1.8-and 2.25-Å resolutions, respectively. The structure shows the typical class I PDZ interaction of PDZ-peptide complex with the consensus sequence -X-(Thr/Ser)-X-Leu. In addition, Asp-634 within the Shank1 PDZ domain recognizes the positively charged Arg at ؊1 position and hydrogen bonds, and salt bridges between Arg-607 and the side chains of the ligand at ؊3 and ؊5 positions contribute further to the recognition of the peptide ligand. Remarkably, whether free or complexed, Shank1 PDZ domains form dimers with a conserved ␤B/␤C loop and N-terminal ␤A strands, suggesting a novel model of PDZ-PDZ homodimerization. This implies that antiparallel dimerization through the N-terminal ␤A strands could be a common configuration among PDZ dimers. Within the dimeric structure, the two-peptide binding sites are arranged so that the N termini of the bound peptide ligands are in close proximity and oriented toward the 2-fold axis of the dimer. This configuration may provide a means of facilitating dimeric organization of PDZ-target assemblies.Multidomain Shank, proline-rich synapse-associated protein, and somatostatin receptor-interacting protein scaffold proteins bind to various membrane and cytoplasmic proteins within the PSDs 1 in excitatory synapses (1, 2). It has been suggested that Shank links N-methyl-D-aspartate receptor-PSD-95 complexes to the actin cytoskeleton, thereby playing a critical role in the organization of cytoskeletal signaling complexes at excitatory synapses (1, 2). The three known members of the Shank family (Shank1-3) all contain multiple sites for alternative splicing and show distinct tissue distributions (2). Although shank proteins vary in molecular mass, they share a common domain organization consisting of seven N-terminal ankyrin repeats followed by an SH3 domain, a PDZ domain, a long proline-rich region, and a SAM domain. All of these motifs are potentially involved in protein-protein interactions. For instance, the proline-rich region commonly acts as a binding site for SH3, EVH1, and WW domains and SAM domains can bind to each other in homomeric and heteromeric fashion, enabling oligomerization of Shank and its interacting proteins (3).PDZs are globular domains containing ϳ80 -100 amino acids (4). The Shank PDZ domain is a class I PDZ recognizing the C-terminal sequence X-(Thr/Ser)-X-Leu (where X represents any amino acid), which enables it to bind a variety of integral membrane proteins; however, it most specifically binds to ...

PDZ domains bind to short segments within target proteins in a sequence-specific fashion. Glutamate receptor-interacting protein (GRIP)/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-␣. In addition the PDZ456 domain mediates the formation of homo-and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-␣ at resolutions of 1.5 and 1.8 Å, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at ␣B5 rather than conserved Leu-732 at ␣B1 makes a direct hydrophobic contact with the side chain of the Tyr at the ؊2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix ␣B, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins.Synaptic localization and clustering of ion channels and receptors is often mediated by scaffolding molecules containing the protein-protein interaction motifs called PDZ (Postsynaptic density-95/Discs large/Zona occludens-1) domains (1). One of the most abundant molecular recognition elements, these globular domains each contain two ␣-helices and six ␤-strands. They usually bind selectively to the C terminus or a short internal segment of interacting proteins (1) and are categorized into four classes according to their specificity for the C-terminal target sequences (2). Class I PDZ domains bind to a C-terminal motif with the sequence X-Ser/Thr-X-Val/Leu-COOH, where X represents any residue, while class II PDZ domains prefer X-⌽-X-⌽-COOH, where ⌽ is usually a large hydrophobic residue. Both class I and II domains have a preference for a hydrophobic residue at the 0 position of the ligand. Class III PDZ domains prefer the sequence X-Asp-X-Val-COOH in which a negatively charged amino acid is at the Ϫ2 position (3), while class IV domains prefer the sequence X-⌿-Asp/Glu-COOH in which an acidic residue is at the C-terminal position and where ⌿ represents an aromatic residue (4). In addition, there are other classes of PDZ domains that do not fall into any of the aforementioned classes (5, 6), and there are minor discrepancies in the proposed classifications of PDZ domains (7,8).Members of the GRIP 1 family proteins (GRIP1 and GRIP2/ ABP) contain six to seven PDZ domains (9, 10, 11). GRIP PDZ45, which is classified as a class II PDZ domain (1), binds to the C terminus of the GluR2/3 subunit of AMPA glutamate receptors (9, 10, 12), while GRIP PDZ6, also a class II PDZ domain, interacts with the ...

Motor proteins not actively involved in transporting cargoes should remain inactive at sites of cargo loading to save energy and remain available for loading. KIF1A/ Unc104 is a monomeric kinesin known to dimerize into a processive motor at high protein concentrations. However, the molecular mechanisms underlying monomer stabilization and monomer-to-dimer transition are not well understood. Here, we report an intramolecular interaction in KIF1A between the forkhead-associated (FHA) domain and a coiled-coil domain (CC2) immediately following the FHA domain. Disrupting this interaction by point mutations in the FHA or CC2 domains leads to a dramatic accumulation of KIF1A in the periphery of living cultured neurons and an enhancement of the microtubule (MT) binding and self-multimerization of KIF1A. In addition, point mutations causing rigidity in the predicted flexible hinge disrupt the intramolecular FHA-CC2 interaction and increase MT binding and peripheral accumulation of KIF1A. These results suggest that the intramolecular FHA-CC2 interaction negatively regulates KIF1A activity by inhibiting MT binding and dimerization of KIF1A, and point to a novel role of the FHA domain in the regulation of kinesin motors.

In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum. Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca 2؉ , but also to the active Ca 2؉ release process during excitation-contraction coupling. Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin. Using in vitro binding assay and site-directed mutagenesis, we found that the second intralu- residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca 2؉ release process of excitation-contraction coupling in muscle cells.Ultrastructural and biochemical evidence suggests that a protein complex exists at the junctional SR 1 membrane in cardiac and skeletal muscle to facilitate the Ca 2ϩ release that occurs during muscle contraction (1-4). These include the ryanodine receptor (RyR), triadin, junctin, and calsequestrin, which may associate into a stable complex at the junctional membrane (5). The RyR contains a large cytoplasmic foot region plus a transmembrane domain at the carboxyl-terminal end. The transmembrane domain of RyR has been shown to contain the conduction pore of the Ca 2ϩ release channel (6). According to the hydropathy plot of Takeshima et al. (7), four putative transmembrane segments (TM1 to TM4) are predicted to span the SR membrane, with residues connecting TM1 and TM2 forming the first intraluminal loop (aa 4581-4640) and residues connecting TM3 and TM4 contributing to the second intraluminal loop (aa 4860 -4917). Elegant studies from other investigators have shown that the second intraluminal loop of RyR participates in the overall ion conduction and selectivity process of the Ca 2ϩ release channel (8 -10). Calsequestrin is the high capacity Ca 2ϩ storage protein located in the lumen of the SR (11). In striated muscles, calsequestrins are largely tethered to the terminal cisternae of SR, leading to the suggestion that they could sequester Ca 2ϩ to sites of Ca 2ϩ release (12). Triadin and junctin are structurally similar integral membrane proteins having a short aminoterminal cytoplasmic domain and a long stretch exposed to the luminal side of the SR (13,14). The luminal region is especially enriched in multiple clusters of alternating lysine and glutamic acid residues, named as the KEKE motif (14 -16).Previous studies show that triadin could directly modulate the activity of the RyR1 channel in a reconstitutional system (17, 18). Calsequestrin has been proposed to indirectly regulate the RyR function presumably through its interaction with triadin and junctin (5,19). Recently, the binding regions of calsequestrin and the histidine-rich Ca 2ϩ -binding protein to triadin were identified in our group (20,21). However, the nature of interaction between triadin ...

ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (MjLon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-Å resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the ␤4-␣2 segment is an ␣-helix, whereas it is a ␤-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pK a of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.In all cells, energy-dependent proteolysis plays a key role in the rapid turnover of short-lived regulatory proteins and in the elimination of defective and denatured proteins (1). Bacterial cells possess a number of ATP-dependent proteases, which are complex enzymes containing both ATPase and proteolytic activity as separate domains within a single polypeptide or as individual subunits within complex assemblies. Escherichia coli, for example, express five different ATP-dependent proteases: Lon, ClpAP, ClpXP, HslUV (ClpYQ), and FtSH (2). Homologous proteases have also been identified in archaea and eubacteria, as well as in numerous eukaryotes. Some archaeal Lons have one or two putative transmembrane regions, suggesting that they are membrane-associated (3). The proteolytic components of ATP-dependent proteases include several different types of active sites. For instance, ClpP is a classical serine protease (4), whereas HslV has a catalytic N-terminal Thr residue (5).Lon was the first ATP-dependent protease to be described (6). Similar to the molecular chaperon, Lon recognizes a broad range of proteins and mediates their turnover of abnormal and short-lived normal proteins. Indeed, through degradation of various specialized proteins, Lon is involved in the regulation of a number of biological functions (7). Moreover, it also reportedly acts as a DNA-binding protein, influencing the regulation of DNA replication and gene...

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.

It has been suggested that the large conductance Ca 2؉ -activated K ؉ channel contains one or more domains known as regulators of K ؉ conductance (RCK) in its cytosolic C terminus. Here, we show that the second RCK domain (RCK2) is functionally important and that it forms a heterodimer with RCK1 via a hydrophobic interface. Mutant channels lacking RCK2 are nonfunctional despite their tetramerization and surface expression. The hydrophobic residues that are expected to form an interface between RCK1 and RCK2, based on the crystal structure of the bacterial MthK channel, are well conserved, and the interactions of these residues were confirmed by mutant cycle analysis. The hydrophobic interaction appears to be critical for the Ca 2؉ -dependent gating of the large conductance Ca 2؉ -activated K ؉ channel.Large conductance calcium-activated potassium (BK Ca ) 2 channels play a key role in modulating a number of important physiological processes, such as neuronal excitability, frequency tuning of hair cells, smooth muscle contraction, and immunity (1-9). BK Ca channels are activated by membrane depolarization and an increase in intracellular calcium (10 -13). Thus, BK Ca channels are considered to be molecular integrators of biochemical and electrical signals. Membrane depolarization and calcium binding activate BK Ca channels independently via separate regions of the ␣ subunit of the channel (Slo). The transmembrane segments of the Slo channel, S1-S6, are structurally similar to those of voltage-gated potassium channels, and as in these channels, charged residues in the Slo S1-S4 segments are thought to be involved in the voltage-dependent gating of the channel (14 -20). It is generally accepted that the bulkycytoplasmicCterminusofSloisresponsibleforthecalciumdependent activation of the channel (21-23).The cytoplasmic C terminus of Slo has been proposed to contain more than two Ca 2ϩ -sensing sites, a high affinity site called the Ca 2ϩ bowl, a low affinity site, and additional high affinity sites within a structural module known as the regulator of K ϩ conductance (RCK) domain (22, 24 -29). The Ca 2ϩ bowl is composed of a series of Asp residues and binds Ca 2ϩ with micromolar affinity. Mutations here have been shown to cause positive shifts in the conductance-voltage (G-V) relationship at constant [Ca 2ϩ ], which are similar to those observed with the wild-type channel when [Ca 2ϩ ] is lowered (22,23,26,30,31). The RCK domain is found primarily in prokaryotic ligandgated K ϩ channels and in some bacterial K ϩ uptake and efflux systems, in which it is also called the K ϩ transport nucleotidebinding (KTN) domain (25,(32)(33)(34). Crystal structures of RCK domains have been determined from Ca 2ϩ -activated K ϩ channels in Escherichia coli and Methanobacterium thermoautotrophicum (24,25,35,36). The structure of the tetrameric MthK channel shows that an octameric complex could be formed by intermolecular interactions on fixed and flexible interfaces between a tetramer of dimeric RCK domains. This complex, called the...