Crystal structure of the Legionella pneumophila Lpg2936 in complex with the cofactor S‐adenosyl‐L‐methionine reveals novel insights into the mechanism of RsmE family methyltransferases

The methylation of U1498 located in the 16S ribosomal RNA of Escherichia coli is an important modification affecting ribosomal activity. RsmE methyltransferases methylate specifically this position in a mechanism that requires an S‐adenosyl‐L‐methionine (AdoMet) molecule as cofactor. Here we report the structure of Apo and AdoMet‐bound Lpg2936 from Legionella pneumophila at 1.5 and 2.3 Å, respectively. The protein comprises an N‐terminal PUA domain and a C‐terminal SPOUT domain. The latter is responsible for protein dimerization and cofactor binding. Comparison with similar structures suggests that Lpg2936 is an RsmE‐like enzyme that can target the equivalent of U1498 in the L. pneumophila ribosomal RNA, thereby potentially enhancing ribosomal activity during infection‐mediated effector production. The multiple copies of the enzyme found in both structures reveal a flexible conformation of the bound AdoMet ligand. Isothermal titration calorimetry measurements suggest an asymmetric two site binding mode. Our results therefore also provide unprecedented insights into AdoMet/RsmE interaction, furthering our understanding of the RsmE catalytic mechanism.     

Anchorage‐dependent binding of integrin I‐domain to adhesion ligands

The dynamic interactions between leukocyte integrin receptors and ligands in the vascular endothelium, extracellular matrix, or invading pathogens result in leukocyte adhesion, extravasation, and phagocytosis. This work examined the mechanical strength of the connection between iC3b, a complement component that stimulates phagocytosis, and the ligand‐binding domain, the I‐domain, of integrin α_Mβ₂. Single‐molecule force measurements of α_M I‐domain–iC3b complexes were conducted by atomic force microscope. Strikingly, depending on loading rates, immobilization of the I‐domain via its C‐terminus resulted in a 1.3‐fold to 1.5‐fold increase in unbinding force compared with I‐domains immobilized via the N‐terminus. The force spectra (unbinding force versus loading rate) of the I‐domain–iC3b complexes revealed that the enhanced mechanical strength is due to a 2.4‐fold increase in the lifetime of the I‐domain–iC3b bond. Given the structural and functional similarity of all integrin I‐domains, our result supports the existing allosteric regulatory model by which the ligand binding strength of integrin can be increased rapidly when a force is allowed to stretch the C‐terminus of the I‐domain. This type of mechanism may account for the rapid ligand affinity adjustment during leukocyte migration. Copyright © 2015 John Wiley & Sons, Ltd.     

Structure of the frequency‐interacting RNA helicase: a protein interaction hub for the circadian clock

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ‐interacting RNA Helicase (FRH) rhythmically represses gene expression by the white‐collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ‐interacting region at the FRH N‐terminus has variable structure in the absence of FRQ. A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ. A V142G substitution near the N‐terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N‐terminus and KOW module.     

The dense‐core vesicle maturation protein CCCP‐1 binds RAB‐2 and membranes through its C‐terminal domain

Dense‐core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled‐coil protein CCCP‐1 was identified as a component of the DCV biogenesis pathway in the nematode Caenorhabditis elegans. CCCP‐1 binds the small GTPase RAB‐2 and colocalizes with it at the trans‐Golgi. Here, we report a structure‐function analysis of CCCP‐1 to identify domains of the protein important for its localization, binding to RAB‐2, and function in DCV biogenesis. We find that the CCCP‐1 C‐terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP‐1 localization and for binding to RAB‐2, and is required for the function of CCCP‐1 in DCV biogenesis. In addition, CCCP‐1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid‐binding motif. We conclude that CCCP‐1 is a coiled‐coil protein that binds an activated Rab and localizes to the Golgi via its C‐terminus, properties similar to members of the golgin family of proteins. CCCP‐1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.      

Crystal structure of Streptococcus pneumoniae Sp1610, a putative tRNA methyltransferase,in complex with S‐adenosyl‐L‐methionine

Streptococcus pneumoniae Sp1610, a Class‐I fold S‐adenosylmethionine (AdoMet)‐dependent methyltransferase, is a member of the COG2384 family in the Clusters of Orthologous Groups database, which catalyzes the methylation of N¹‐adenosine at position 22 of bacterial tRNA. We determined the crystal structure of Sp1610 in the ligand‐free and the AdoMet‐bound forms at resolutions of 2.0 and 3.0 Å, respectively. The protein is organized into two structural domains: the N‐terminal catalytic domain with a Class I AdoMet‐dependent methyltransferase fold, and the C‐terminal substrate recognition domain with a novel fold of four α‐helices. Observations of the electrostatic potential surface revealed that the concave surface located near the AdoMet binding pocket was predominantly positively charged, and thus this was predicted to be an RNA binding area. Based on the results of sequence alignment and structural analysis, the putative catalytic residues responsible for substrate recognition are also proposed.     

Structural insights into the T6SS effector protein Tse3 and the Tse3–Tsi3 complex from Pseudomonas aeruginosa reveal a calcium‐dependent membrane‐binding mechanism

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm‐localized immunity protein Tsi3 to prevent potential self‐intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3–Tsi3 complex. Tse3 contains an annexin repeat‐like fold at the N‐terminus and a G‐type lysozyme fold at the C‐terminus. One loop in the N‐terminal domain (Loop 12) and one helix (α9) from the C‐terminal domain together anchor Tse3 and the Tse3–Tsi3 complex to membrane in a calcium‐dependent manner in vitro, and this membrane‐binding ability is essential for Tse3's activity. In the C‐terminal domain, a Y‐shaped groove present on the surface likely serves as the PG binding site. Two calcium‐binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3–Tsi3 structure, three loops of Tsi3 insert into the substrate‐binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3–Tsi3 complex.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

Structural basis for cellulose binding by the type A carbohydrate‐binding module 64 of Spirochaeta thermophila

Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate‐binding module 64 (CBM64) appended at the C‐terminus. CBM64 adsorbs to various β1‐4‐linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly‐roll‐like fold corresponding to a surface‐binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of β‐linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855–858. © 2016 Wiley Periodicals, Inc.     

Structure of full‐length class I chitinase from rice revealed by X‐ray crystallography and small‐angle X‐ray scattering

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht‐2, is composed of an N‐terminal chitin‐binding domain (ChBD) and a C‐terminal catalytic domain (CatD), which are connected by a proline‐ and threonine‐rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full‐length structure of OsChia1b was analyzed by X‐ray crystallography and small‐angle X‐ray scattering (SAXS). We determined the crystal structure of full‐length OsChia1b at 2.00‐Å resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri‐N‐acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two‐domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline α‐chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of α‐chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010. © 2010 Wiley‐Liss,Inc.     

Analysis of the Binding Moiety Mediating the Interaction between Monocarboxylate Transporters and Carbonic Anhydrase II

Proton-coupled monocarboxylate transporters (MCTs) mediate the exchange of high energy metabolites like lactate between different cells and tissues. We have reported previously that carbonic anhydrase II augments transport activity of MCT1 and MCT4 by a noncatalytic mechanism, while leaving transport activity of MCT2 unaltered. In the present study, we combined electrophysiological measurements in Xenopus oocytes and pulldown experiments to analyze the direct interaction between carbonic anhydrase II (CAII) and MCT1, MCT2, and MCT4, respectively. Transport activity of MCT2-WT, which lacks a putative CAII-binding site, is not augmented by CAII. However, introduction of a CAII-binding site into the C terminus of MCT2 resulted in CAII-mediated facilitation of MCT2 transport activity. Interestingly, introduction of three glutamic acid residues alone was not sufficient to establish a direct interaction between MCT2 and CAII, but the cluster had to be arranged in a fashion that allowed access to the binding moiety in CAII. We further demonstrate that functional interaction between MCT4 and CAII requires direct binding of the enzyme to the acidic cluster ⁴³¹EEE in the C terminus of MCT4 in a similar fashion as previously shown for binding of CAII to the cluster ⁴⁸⁹EEE in the C terminus of MCT1. In CAII, binding to MCT1 and MCT4 is mediated by a histidine residue at position 64. Taken together, our results suggest that facilitation of MCT transport activity by CAII requires direct binding between histidine 64 in CAII and a cluster of glutamic acid residues in the C terminus of the transporter that has to be positioned in surroundings that allow access to CAII.     

Architecture and self‐assembly of the SARS‐CoV‐2 nucleocapsid protein

The COVID‐2019 pandemic is the most severe acute public health threat of the twenty‐first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS‐CoV‐2 coronavirus. Here, we examine the architecture and self‐assembly properties of the SARS‐CoV‐2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS‐CoV. While the N2b domain forms a dimer in solution, addition of the C‐terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen‐deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α‐helix that self‐associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS‐CoV‐2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self‐assembly properties of a key protein in the SARS‐CoV‐2 life cycle, with implications for both drug design and antibody‐based testing.     

Disulfide bond of Mycoplasma pneumoniae community‐acquired respiratory distress syndrome toxin is essential to maintain the ADP‐ribosylating and vacuolating activities

Mycoplasma pneumoniae is the leading cause of bacterial community‐acquired pneumonia among hospitalised children in United States and worldwide. Community‐acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N‐terminus of CARDS toxin exhibits ADP‐ribosyltransferase (ADPRT) activity, and the C‐terminus possesses binding and vacuolating activities. Thiol‐trapping experiments of wild‐type (WT) and cysteine‐to‐serine‐mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin‐mediated ADPRT activity‐associated IL‐1β production in U937 cells and the recovery of vacuolating activity in the protease‐released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects.     

DbpA is a region‐specific RNA helicase

DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp.     

Germinal‐center kinase‐like kinase co‐crystal structure reveals a swapped activation loop and C‐terminal extension

Germinal‐center kinase‐like kinase (GLK, Map4k3), a GCK‐I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation‐loop swapped dimer with more than 20 amino acids of ordered density at the carboxy‐terminus. This C‐terminal PEST region binds intermolecularly to the hydrophobic groove of the N‐terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an “active” kinase, including the salt bridge between the C‐helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P‐loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.     

Structure of the Shq1-Cbf5-Nop10-Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita

Shq1 is a conserved protein required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs), including human telomerase. We report the structure of the Shq1-specific domain alone and in complex with H/ACA RNP proteins Cbf5, Nop10 and Gar1. The Shq1-specific domain adopts a novel helical fold and primarily contacts the PUA domain and the otherwise disordered C-terminal extension (CTE) of Cbf5. The structure shows that dyskeratosis congenita mutations found in the CTE of human Cbf5 likely interfere with Shq1 binding. However, most mutations in the PUA domain are not located at the Shq1-binding surface and also have little effect on the yeast Cbf5-Shq1 interaction. Shq1 binds Cbf5 independently of the H/ACA RNP proteins Nop10, Gar1 and Nhp2 and the assembly factor Naf1, but shares an overlapping binding surface with H/ACA RNA. Shq1 point mutations that disrupt Cbf5 interaction suppress yeast growth particularly at elevated temperatures. Our results suggest that Shq1 functions as an assembly chaperone that protects the Cbf5 protein complexes from non-specific RNA binding and aggregation before assembly of H/ACA RNA.     

The Ras‐binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins

RGS14 is a 60 kDa protein that contains a regulator of G protein signaling (RGS) domain near its N‐terminus, a central region containing a pair of tandem Ras‐binding domains (RBD), and a GPSM (G protein signaling modulator) domain (a.k.a. Gi/o‐Loco binding [GoLoco] motif) near its C‐terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full‐length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter‐domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR‐activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation. J. Cell. Biochem. 114: 1414–1423, 2013. © 2012 Wiley Periodicals, Inc.     

Critical role of the N‐terminal cyclic AMP‐binding domain of Epac2 in its subcellular localization and function

cAMP is a well‐known regulator of exocytosis, and cAMP‐GEFII (Epac2) is involved in the potentiation of cAMP‐dependent, PKA‐independent regulated exocytosis in secretory cells. However, the mechanisms of its action are not fully understood. In the course of our study of Epac2 knockout mice, we identified a novel splicing variant of Epac2, which we designate Epac2B, while renaming the previously identified Epac2 Epac2A. Epac2B, which lacks the first cAMP‐binding domain A in the N‐terminus but has the second cAMP‐binding domain B of Epac2A, possesses GEF activity towards Rap1, as was found for Epac2A. Immunocytochemical analysis revealed that exogenously introduced Epac2A into insulin‐secreting MIN6 cells was localized near the plasma membrane, while Epac2B was found primarily in the cytoplasm. Interestingly, cAMP‐binding domain A alone introduced into MIN6 cells was also localized near the plasma membrane. In MIN6 cells, Epac2A was involved in triggering hormone secretion by stimulation with 5.6 mM glucose plus 1 mM 8‐Bromo‐cAMP, but Epac2B was not. The addition of a membrane‐targeting signal to the N‐terminus of Epac2B was able to mimic the effect of Epac2A on hormone secretion. Thus, the present study indicates that the N‐terminal cAMP‐binding domain A of Epac2A plays a critical role in determining its subcellular localization and potentiating insulin secretion by cAMP. J. Cell. Physiol. 219: 652–658, 2009. © 2009 Wiley‐Liss, Inc.     

Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu⁺‐ATPase CopA from Archaeoglobus fulgidus belongs to the P_1B family of the P‐type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P_1B‐1‐type ATPases is the presence of soluble metal binding domains at the N‐terminus (N‐MBDs). The N‐MBDs exhibit a conserved ferredoxin‐like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N‐MBDs enable Cu⁺ regulation of turnover rates apparently through Cu‐sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N‐terminal MBD and a C‐terminal MBD (C‐MBD). The functional role of the unique C‐MBD has not been established. Here, we report the crystal structure of the apo, oxidized C‐MBD to 2.0 Å resolution. In the structure, two C‐MBD monomers form a domain‐swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C‐MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A‐domain), has been investigated. Interestingly, the C‐MBD interacts specifically with both of these domains, independent of the presence of Cu⁺ or nucleotides. These data reinforce the uniqueness of the C‐MBD and suggest a distinct structural role for the C‐MBD in CopA transport. Proteins 2010. © 2010 Wiley‐Liss, Inc.