A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the ß subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

Structural basis of ubiquitin recognition by the winged-helix domain of Cockayne syndrome group B protein.

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.

Structural basis for methyl-donor-dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD.

The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA.

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA.

In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA(Ile)2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site.

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor.

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.

Structural insights into selective interaction between type IIa receptor protein tyrosine phosphatases and Liprin-α.

Synapse formation is induced by transsynaptic interaction of neuronal cell-adhesion molecules termed synaptic organizers. Type IIa receptor protein tyrosine phosphatases (IIa RPTPs) function as presynaptic organizers. The cytoplasmic domain of IIa RPTPs consists of two phosphatase domains, and the membrane-distal one (D2) is essential for synapse formation. Liprin-α, which is an active zone protein critical for synapse formation, interacts with D2 via its C-terminal domain composed of three tandem sterile alpha motifs (tSAM). Structural mechanisms of this critical interaction for synapse formation remain elusive. Here, we report the crystal structure of the complex between mouse PTPδ D2 and Liprin-α3 tSAM at 1.91 Å resolution. PTPδ D2 interacts with the N-terminal helix and the first and second SAMs (SAM1 and SAM2, respectively) of Liprin-α3. Structure-based mutational analyses in vitro and in cellulo demonstrate that the interactions with Liprin-α SAM1 and SAM2 are essential for the binding and synaptogenic activity.

Structural basis of guanine nucleotide exchange for Rab11 by SH3BP5.

The Rab GTPase family is a major regulator of membrane traffic in eukaryotic cells. The Rab11 subfamily plays important roles in specific trafficking events such as exocytosis, endosomal recycling, and cytokinesis. SH3BP5 and SH3BP5-like (SH3BP5L) proteins have recently been found to serve as guanine nucleotide exchange factors (GEF) for Rab11. Here, we report the crystal structures of the SH3BP5 GEF domain alone and its complex with Rab11a. SH3BP5 exhibits a V-shaped structure comprising two coiled coils. The coiled coil composed of α1, and α4 is solely responsible for the Rab11a binding and GEF activity. SH3BP5 pulls out and deforms switch I of Rab11a so as to facilitate the GDP release from Rab11a. SH3BP5 interacts with the N-terminal region, switch I, interswitch, and switch II of Rab11a. SH3BP5 and SH3BP5L localize to Rab11-positive recycling endosomes and show GEF activity for all of the Rab11 family but not for Rab14. Fluorescence-based GEF assays combined with site-directed mutagenesis reveal the essential interactions between SH3BP5 and Rab11 family proteins for the GEF reaction on recycling endosomes.

Crystal structure of archaeal tRNA(m(1)G37)methyltransferase aTrm5.

Methylation of the N1 atom of guanosine at position 37 in tRNA, the position 3'-adjacent to the anticodon, generates the modified nucleoside m(1)G37. In archaea and eukaryotes, m(1)G37 synthesis is catalyzed by tRNA(m(1)G37)methyltransferase (archaeal or eukaryotic Trm5, a/eTrm5). Here we report the crystal structure of archaeal Trm5 (aTrm5) from Methanocaldococcus jannaschii (formerly known as Methanococcus jannaschii) in complex with the methyl donor analogue at 2.2 A resolution. The crystal structure revealed that the entire protein is composed of three structural domains, D1, D2, and D3. In the a/eTrm5 primary structures, D2 and D3 are highly conserved, while D1 is not conserved. The D3 structure is the Rossmann fold, which is the hallmark of the canonical class-I methyltransferases. The a/eTrm5-defining domain, D2, exhibits structural similarity to some class-I methyltransferases. In contrast, a DALI search with the D1 structure yielded no structural homologues. In the crystal structure, D3 contacts both D1 and D2. The residues involved in the D1:D3 interactions are not conserved, while those participating in the D2:D3 interactions are well conserved. D1 and D2 do not contact each other, and the linker between them is disordered. aTrm5 fragments corresponding to the D1 and D2-D3 regions were prepared in a soluble form. The NMR analysis of the D1 fragment revealed that D1 is well folded by itself, and it did not interact with either the D2-D3 fragment or the tRNA. The NMR analysis of the D2-D3 fragment revealed that it is well folded, independently of D1, and that it interacts with tRNA. Furthermore, the D2-D3 fragment was as active as the full-length enzyme for tRNA methylation. The positive charges on the surface of D2-D3 may be involved in tRNA binding. Therefore, these findings suggest that the interaction between D1 and D3 is not persistent, and that the D2-D3 region plays the major role in tRNA methylation.

Structural insights into modulation and selectivity of transsynaptic neurexin-LRRTM interaction.

Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). Here, we report the crystal structure of the Nrxn1ß-LRRTM2 complex at 3.4 Å resolution. The Nrxn1ß-LRRTM2 interface involves Ca2+-mediated interactions and overlaps with the Nrxn-neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn.

Structural insights into ubiquitin phosphorylation by PINK1.

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.

Structural insights into two distinct binding modules for Lys63-linked polyubiquitin chains in RNF168.

The E3 ubiquitin (Ub) ligase RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs). The recruitment of RNF168 by ubiquitylated targets involves two distinct regions, Ub-dependent DSB recruitment module (UDM) 1 and UDM2. Here we report the crystal structures of the complex between UDM1 and Lys63-linked diUb (K63-Ub2) and that between the C-terminally truncated UDM2 (UDM2ΔC) and K63-Ub2. In both structures, UDM1 and UDM2ΔC fold as a single α-helix. Their simultaneous bindings to the distal and proximal Ub moieties provide specificity for Lys63-linked Ub chains. Structural and biochemical analyses of UDM1 elucidate an Ub-binding mechanism between UDM1 and polyubiquitylated targets. Mutations of Ub-interacting residues in UDM2 prevent the accumulation of RNF168 to DSB sites in U2OS cells, whereas those in UDM1 have little effect, suggesting that the interaction of UDM2 with ubiquitylated and polyubiquitylated targets mainly contributes to the RNF168 recruitment.

Structural basis of trans-synaptic interactions between PTPδ and SALMs for inducing synapse formation.

Synapse formation is triggered by trans-synaptic interactions of cell adhesion molecules, termed synaptic organizers. Three members of type-II receptor protein tyrosine phosphatases (classified as type-IIa RPTPs; PTPδ, PTPσ and LAR) are known as presynaptic organizers. Synaptic adhesion-like molecules (SALMs) have recently emerged as a family of postsynaptic organizers. Although all five SALM isoforms can bind to the type-IIa RPTPs, only SALM3 and SALM5 reportedly have synaptogenic activities depending on their binding. Here, we report the crystal structures of apo-SALM5, and PTPδ-SALM2 and PTPδ-SALM5 complexes. The leucine-rich repeat (LRR) domains of SALMs interact with the second immunoglobulin-like (Ig) domain of PTPδ, whereas the Ig domains of SALMs interact with both the second and third Ig domains of PTPδ. Unexpectedly, the structures exhibit the LRR-mediated 2:2 complex. Our synaptogenic co-culture assay using site-directed SALM5 mutants demonstrates that presynaptic differentiation induced by PTPδ-SALM5 requires the dimeric property of SALM5.

Structural basis of epilepsy-related ligand-receptor complex LGI1-ADAM22.

Epilepsy is a common brain disorder throughout history. Epilepsy-related ligand-receptor complex, LGI1-ADAM22, regulates synaptic transmission and has emerged as a determinant of brain excitability, as their mutations and acquired LGI1 autoantibodies cause epileptic disorders in human. Here, we report the crystal structure of human LGI1-ADAM22 complex, revealing a 2:2 heterotetrameric assembly. The hydrophobic pocket of the C-terminal epitempin-repeat (EPTP) domain of LGI1 binds to the metalloprotease-like domain of ADAM22. The N-terminal leucine-rich repeat and EPTP domains of LGI1 mediate the intermolecular LGI1-LGI1 interaction. A pathogenic R474Q mutation of LGI1, which does not exceptionally affect either the secretion or the ADAM22 binding, is located in the LGI1-LGI1 interface and disrupts the higher-order assembly of the LGI1-ADAM22 complex in vitro and in a mouse model for familial epilepsy. These studies support the notion that the LGI1-ADAM22 complex functions as the trans-synaptic machinery for precise synaptic transmission.

Trm5 and TrmD: Two Enzymes from Distinct Origins Catalyze the Identical tRNA Modification, m¹G37.

The N¹-atom of guanosine at position 37 in transfer RNA (tRNA) is methylated by tRNA methyltransferase 5 (Trm5) in eukaryotes and archaea, and by tRNA methyltransferase D (TrmD) in bacteria. The resultant modified nucleotide m¹G37 positively regulates the aminoacylation of the tRNA, and simultaneously functions to prevent the +1 frameshift on the ribosome. Interestingly, Trm5 and TrmD have completely distinct origins, and therefore bear different tertiary folds. In this review, we describe the different strategies utilized by Trm5 and TrmD to recognize their substrate tRNAs, mainly based on their crystal structures complexed with substrate tRNAs.

Structural basis of the interaction between Topoisomerase IIIß and the TDRD3 auxiliary factor.

Topoisomerase IIIß (TOP3ß) is a DNA/RNA topoisomerase that has been implicated in epigenetic or translational control of gene expression. In cells, TOP3ß co-exists with its specific auxiliary factor, TDRD3. TDRD3 serves as a scaffold protein to recruit TOP3ß to its DNA/RNA substrates accumulating in specific cellular sites such as methylated chromatins or neural stress granules. Here we report the crystal structures of the catalytic domain of TOP3ß, the DUF1767-OB-fold domains of TDRD3 and their complex at 3.44 Å, 1.62 Å and 3.6 Å resolutions, respectively. The toroidal-shaped catalytic domain of TOP3ß binds the OB-fold domain of TDRD3. The TDRD3 OB-fold domain harbors the insertion loop, which is protruding from the core structure. Both the insertion loop and core region interact with TOP3ß. Our pull-down binding assays showed that hydrophobic characters of the core surface and the amino- and carboxy-terminal regions of the insertion loop are essential for the interaction. Furthermore, by comparison with the structure of the homologous Topoisomerase IIIα (TOP3α)-RMI1 complex, we identified Arg96, Val109, Phe139 and the short insertion loop of TDRD3 as the critical structural elements for the specific interaction with TOP3ß to avoid the non-cognate interaction with TOP3α.

Crystal structure of Sec10, a subunit of the exocyst complex.

The exocyst complex is a heterooctameric protein complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. This complex plays an essential role in trafficking secretory vesicles to the plasma membrane through its interaction with phosphatidylinositol 4,5-bisphosphate and small GTPases. To date, the near-full-length structural information of each subunit has been limited to Exo70, although the C-terminal half structures of Sec6, Sec15 and Exo84 and the structures of the small GTPase-binding domains of Sec3, Sec5 and Exo84 have been reported. Here, we report the crystal structure of the near-full-length zebrafish Sec10 (zSec10) at 2.73 Å resolution. The structure of zSec10 consists of tandem antiparallel helix bundles that form a straight rod, like helical core regions of other exocyst subunits. This structure provides the first atomic details of Sec10, which may be useful for future functional and structural studies of this subunit and the exocyst complex.

Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30.

Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub2) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.

Mechanisms of splicing-dependent trans-synaptic adhesion by PTPδ-IL1RAPL1/IL-1RAcP for synaptic differentiation.

Synapse formation is triggered through trans-synaptic interaction between pairs of pre- and postsynaptic adhesion molecules, the specificity of which depends on splice inserts known as 'splice-insert signaling codes'. Receptor protein tyrosine phosphatase δ (PTPδ) can bidirectionally induce pre- and postsynaptic differentiation of neurons by trans-synaptically binding to interleukin-1 receptor accessory protein (IL-1RAcP) and IL-1RAcP-like-1 (IL1RAPL1) in a splicing-dependent manner. Here, we report crystal structures of PTPδ in complex with IL1RAPL1 and IL-1RAcP. The first immunoglobulin-like (Ig) domain of IL1RAPL1 directly recognizes the first splice insert, which is critical for binding to IL1RAPL1. The second splice insert functions as an adjustable linker that positions the Ig2 and Ig3 domains of PTPδ for simultaneously interacting with the Ig1 domain of IL1RAPL1 or IL-1RAcP. We further identified the IL1RAPL1-specific interaction, which appears coupled to the first-splice-insert-mediated interaction. Our results thus reveal the decoding mechanism of splice-insert signaling codes for synaptic differentiation induced by trans-synaptic adhesion between PTPδ and IL1RAPL1/IL-1RAcP.

Structure of Slitrk2-PTPδ complex reveals mechanisms for splicing-dependent trans-synaptic adhesion.

Selective binding between pre- and postsynaptic adhesion molecules can induce synaptic differentiation. Here we report the crystal structure of a synaptogenic trans-synaptic adhesion complex between Slit and Trk-like family member 2 (Slitrk2) and receptor protein tyrosine phosphatase (RPTP) δ. The structure and site-directed mutational analysis revealed the structural basis of splicing-dependent adhesion between Slitrks and type IIa RPTPs for inducing synaptic differentiation.