General Binding Mode Research Articles

Mechanistic Insights into the Interactions of Arl8b with the RUN Domains of PLEKHM1 and SKIP

Arl8b, a specific Arf-like family GTPase present on lysosome, and plays critical roles in many lysosome-related cellular processes such as autophagy. The active Arl8b can be specifically recognized by the RUN domains of two Arl8b-effectors PLEKHM1 and SKIP, thereby regulating the autophagosome/lysosome membrane fusion and the intracellular lysosome positioning, respectively. However, the mechanistic bases underlying the interactions of Arl8b with the RUN domains of PLEKHM1 and SKIP remain elusive. Here, we report the two high-resolution crystal structures of the active Arl8b in complex with the RUN domains of PLEKHM1 and SKIP. In addition to elucidating the detailed molecular mechanism governing the specific interactions of the active Arl8b with the RUN domains of PLEKHM1 and SKIP, the determined complex structures also reveal a general binding mode shared by the PLEKHM1 and SKIP RUN domains for interacting with the active Arl8b. Furthermore, we uncovered a competitive relationship between the RUN domains of PLEKHM1 and SKIP in binding to the active Arl8b as well as a unique small GTPase-binding mode adopted by the PLEKHM1 and SKIP RUN domains, thereby enriching the repertoire of the RUN domain/small GTPase interaction modes. In all, our findings provide new mechanistic insights into the interactions of the active Arl8b with PLEKHM1 and SKIP, and are valuable for further understanding the working modes of these proteins in relevant cellular processes.

General anesthetic binding mode via hydration with weak affinity and molecular discrimination: General anesthetic dissolution in interfacial water of the common binding site of GABAA receptor.

The GABAA receptor (GABAAR) is a target channel for the loss of awareness of general anesthesia. General anesthetic (GA) spans a wide range of chemical structures, such as monatomic molecules, barbital acids, phenols, ethers, and alkanes. GA has a weak binding affinity, and the affinity has a characteristic that correlates with the solubility in olive oil rather than the molecular shape. The GA binding site of GABAAR is common to GAs and exists in the transmembrane domain of the GABAAR intersubunit. In this study, the mechanism of GA binding, which allows binding of various GAs with intersubunit selectivity, was elucidated from the hydration analysis of the binding site. Regardless of the diverse GA chemical structures, a strong correlation was observed between the binding free energy and total dehydration number of the binding process. The GA binding free energy was more involved in the binding dehydration and showed molecular recognition that allowed for the binding of various GA structures via binding site hydration. We regarded the GA substitution for the interfacial water molecule of the binding site as a dissolution into the interfacial hydration layer. The elucidation of the GA binding mechanism mediated by hydration at the GABAAR common binding site provides a rationale for the combined use of anesthetics in medical practice and its combination adjustments via drug interactions.

Phosphorylation regulates the binding of autophagy receptors to FIP200 Claw domain for selective autophagy initiation

The ULK complex initiates the autophagosome formation, and has recently been implicated in selective autophagy by interacting with autophagy receptors through its FIP200 subunit. However, the structural mechanism underlying the interactions of autophagy receptors with FIP200 and the relevant regulatory mechanism remain elusive. Here, we discover that the interactions of FIP200 Claw domain with autophagy receptors CCPG1 and Optineurin can be regulated by the phosphorylation in their respective FIP200-binding regions. We determine the crystal structures of FIP200 Claw in complex with the phosphorylated CCPG1 and Optineurin, and elucidate the detailed molecular mechanism governing the interactions of FIP200 Claw with CCPG1 and Optineurin as well as their potential regulations by kinase-mediated phosphorylation. In addition, we define the consensus FIP200 Claw-binding motif, and find other autophagy receptors that contain this motif within their conventional LC3-interacting regions. In all, our findings uncover a general and phosphoregulatable binding mode shared by many autophagy receptors to interact with FIP200 Claw for autophagosome biogenesis, and are valuable for further understanding the molecular mechanism of selective autophagy.

Structural Basis of the Modulation of the Voltage-Gated Calcium Ion Channel Cav 1.1 by Dihydropyridine Compounds*.

1,4‐Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L‐type voltage‐gated Ca2+ (Cav) channels. DHP compounds exhibit chirality‐specific antagonistic or agonistic effects. The structure of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo‐EM structures of nanodisc‐embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)‐(+)‐Bay K8644, and a titration of its agonistic enantiomer (S)‐(−)‐Bay K8644 at resolutions of 2.9–3.4 Å. The amlodipine‐bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)‐(−)‐Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Structural Basis of the Modulation of the Voltage‐Gated Calcium Ion Channel Cav1.1 by Dihydropyridine Compounds**

Abstract1,4‐Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L‐type voltage‐gated Ca2+ (Cav) channels. DHP compounds exhibit chirality‐specific antagonistic or agonistic effects. The structure of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo‐EM structures of nanodisc‐embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)‐(+)‐Bay K8644, and a titration of its agonistic enantiomer (S)‐(−)‐Bay K8644 at resolutions of 2.9–3.4 Å. The amlodipine‐bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)‐(−)‐Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Mechanistic Insights into the Interactions of Ras Subfamily GTPases with the SPN Domain of Autism‐associated SHANK3†

Summary of main observation and conclusionThe active Ras subfamily GTPases, Rap1 and Ras, can be specifically recognized by the SPN domain of SHANK3, mutations of which are associated with many neuropsychiatric diseases such as autism spectrum disorder (ASD). However, the mechanistic bases underlying the interactions of SHANK3 SPN and those Ras subfamily proteins are still elusive. Here, we reported the crystal structures of SHANK3 SPN in complex with the GTP‐bound Rap1b and the Ras‐mimetic Rap1b E30D/K31E double mutant. In addition to uncovering the detailed molecular mechanism governing the specific interactions of SHANK3 SPN with those Ras subfamily proteins, the determined structures also reveal a general binding mode between SHANK3 SPN and its associated Ras subfamily proteins. Finally, our study also provides mechanistic insights into two ASD‐causing R12C and L68P mutations found in the SPN domain of SHANK3, and expands our understanding of the etiology of neuropsychiatric diseases caused by defective SHANK3.

Mechanistic insights into the interactions of NAP1 with the SKICH domains of NDP52 and TAX1BP1

NDP52 and TAX1BP1, two SKIP carboxyl homology (SKICH) domain-containing autophagy receptors, play crucial roles in selective autophagy. The autophagic functions of NDP52 and TAX1BP1 are regulated by TANK-binding kinase 1 (TBK1), which may associate with them through the adaptor NAP1. However, the molecular mechanism governing the interactions of NAP1 with NDP52 and TAX1BP1, as well as the effects induced by TBK1-mediated phosphorylation of NDP52 and TAX1BP1, remains elusive. Here, we report the atomic structures of the SKICH regions of NDP52 and TAX1BP1 in complex with NAP1, which not only uncover the mechanistic bases underpinning the specific interactions of NAP1 with the SKICH domains of NDP52 and TAX1BP1 but also reveal the binding mode of a SKICH domain. Moreover, we uncovered that the SKICH domains of NDP52 and TAX1BP1 share a general binding mode to interact with NAP1. Finally, we also evaluated the currently known TBK1-mediated phosphorylation sites in the SKICH domains of NDP52 and TAX1BP1 on the basis of their interactions with NAP1. In all, our findings provide mechanistic insights into the interactions of NAP1 with NDP52 and TAX1BP1, and are valuable for further understanding the functions of these proteins in selective autophagy.

Conserved Vδ1 Binding Geometry in a Setting of Locus-Disparate pHLA Recognition by δ/αβ T Cell Receptors (TCRs): Insight into Recognition of HIV Peptides by TCRs.

Given the limited set of T cell receptor (TCR) V genes that are used to create TCRs that are reactive to different ligands, such as major histocompatibility complex (MHC) class I, MHC class II, and MHC-like proteins (for example, MIC molecules and CD1 molecules), the Vδ1 segment can be rearranged with Dδ-Jδ-Cδ or Jα-Cα segments to form classical γδTCRs or uncommon αβTCRs using a Vδ1 segment (δ/αβTCR). Here we have determined two complex structures of the δ/αβTCRs (S19-2 and TU55) bound to different locus-disparate MHC class I molecules with HIV peptides (HLA-A*2402-Nef138-10 and HLA-B*3501-Pol448-9). The overall binding modes resemble those of classical αβTCRs but display a strong tilt binding geometry of the Vδ1 domain toward the HLA α1 helix, due to a conserved extensive interaction between the CDR1δ loop and the N-terminal region of the α1 helix (mainly in position 62). The aromatic amino acids of the CDR1δ loop exploit different conformations ("aromatic ladder" or "aromatic hairpin") to accommodate distinct MHC helical scaffolds. This tolerance helps to explain how a particular TCR V region can similarly dock onto multiple MHC molecules and thus may potentially explain the nature of TCR cross-reactivity. In addition, the length of the CDR3δ loop could affect the extent of tilt binding of the Vδ1 domain, and adaptively, the pairing Vβ domains adjust their mass centers to generate differential MHC contacts, hence probably ensuring TCR specificity for a certain peptide-MHC class I (pMHC-I). Our data have provided further structural insights into the TCR recognition of classical pMHC-I molecules, unifying cross-reactivity and specificity.IMPORTANCE The specificity of αβ T cell recognition is determined by the CDR loops of the αβTCR, and the general mode of binding of αβTCRs to pMHC has been established over the last decade. Due to the intrinsic genomic structure of the TCR α/δ chain locus, some Vδ segments can rearrange with the Cα segment, forming a hybrid VδCαVβCβ TCR, the δ/αβTCR. However, the basis for the molecular recognition of such TCRs of their ligands is elusive. Here an αβTCR using the Vδ1 segment, S19-2, was isolated from an HIV-infected patient in an HLA-A*24:02-restricted manner. We then solved the crystal structures of the S19-2 TCR and another δ/αβTCR, TU55, bound to their respective ligands, revealing a conserved Vδ1 binding feature. Further binding kinetics analysis revealed that the S19-2 and TU55 TCRs bind pHLA very tightly and in a long-lasting manner. Our results illustrate the mode of binding of a TCR using the Vδ1 segment to its ligand, virus-derived pHLA.

Structural insights into the interaction and disease mechanism of neurodegenerative disease-associated optineurin and TBK1 proteins.

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients.

The flavonoid luteolin, but not luteolin-7-O-glucoside, prevents a transthyretin mediated toxic response.

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.

Crystal Structure of Human α-2,6 Sialyltransferase

Human β-galactoside α-2,6 sialyltransferase I (ST6Gal-I) establishes the final glycosylation pattern of many glycoproteins by transferring a sialyl moiety to a terminal galactose. Complete sialylation of therapeutic immunoglobulins is essential for their anti-inflammatory activity and for protein stability. However, a complete glycan tree is difficult to achieve in vitro due to limited activity of ST6Gal-I for some galactose acceptors. No structural information on ST6Gal-I that could help to improve the enzymatic properties of ST6Gal-I for biotechnological purposes was previously available. We describe the crystal structure of human ST6Gal-I, which allows rationalizing the inhibitory activity of cytosine-based nucleotides. ST6Gal-I differs from related sialyltransferases by several large insertions and deletions that determine its regio- and substrate specificity. Excitingly, a large glycan binds to the active site in a catalytically competent orientation, representing the general binding mode of any substrate glycoprotein. This binding mode also rationalizes why some galactose acceptors are incompletely sialylated. Comparison with a bacterial sialyltransferase lends first insight into the Michaelis complex. The results support an SN2 mechanism with inversion of configuration at the sialyl residue and suggest substrate-assisted catalysis with a charge relay mechanism that bears conceptual similarity to serine proteases.

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases play important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2, hydroxylation of transcription factors3, and of splicing factor proteins4. Recently, 2OG-oxygenases that catalyze hydroxylation of tRNA5-7 and ribosomal proteins8, have been shown to play roles in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9-12. The finding that the ribosomal oxygenases (ROX) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, ycfD catalyzes arginine-hydroxylation in the ribosomal protein L16; in humans, Mina53 (MYC-induced nuclear antigen) and NO66 (Nucleolar protein 66) catalyze histidine-hydroxylation in ribosomal proteins rpL27a and rpL8, respectively. The functional assignments of the ROX open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in residue- and protein-selectivities of prokaryotic and eukaryotic ROX, crystal structures of ycfD and ycfDRM from E. coli and Rhodothermus marinus with those of human Mina53 and NO66 (hROX) reveal highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-oxygenases. ROX structures in complex with/without their substrates, support their functional assignments as hydroxylases, but not demethylases and reveal how the subfamily has evolved to catalyze the hydroxylation of different residue sidechains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-hydroxylases including the hypoxia-inducible factor asparaginyl-hydroxylase (FIH) and histone Nε-methyl lysine demethylases (KDMs) identifies branchpoints in 2OG-oxygenase evolution and distinguishes between JmjC-hydroxylases and -demethylases catalyzing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate oxidizing species reacts. This coordination flexibility has likely contributed to the evolution of the wide range of reactions catalyzed by iron-oxygenases.

Molecular Insights on the Recognition of a Lactococcus lactis Cell Wall Pellicle by the Phage 1358 Receptor Binding Protein

The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a "shoulder" domain linking the RBP to the rest of the phage and a jelly roll fold "head/host recognition" domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ∼8 Å from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages. Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage 1358 receptor binding protein (RBP) in complex with monosaccharides. The positions and nature of monosaccharides bound to the RBP are in agreement with the pellicle structure and suggest a general binding mode of lactococcal phages to their pellicle saccharidic receptor.

Luminescence of [Ru(bpy)2(dppz)]2+ Bound to RNA Mismatches

The luminescence of rac-[Ru(bpy)2(dppz)](2+) (bpy = 2,2'-bipyridine and dppz = dipyrido[3,2-a:2',3'-c]phenazine) was explored in the presence of RNA oligonucleotides containing a single RNA mismatch (CA and GG) in order to develop a probe for RNA mismatches. While there is minimal luminescence of [Ru(bpy)2(dppz)](2+) in the presence of matched RNA due to weak binding, the luminescence is significantly enhanced in the presence of a single CA mismatch. The luminescence differential between CA mismatched and matched RNA is substantially higher compared to the DNA analogue, and therefore, [Ru(bpy)2(dppz)](2+) appears to be also a sensitive light switch probe for a CA mismatch in duplex RNA. Although the luminescence intensity is lower in the presence of RNA than DNA, Förster resonance energy transfer (FRET) between the donor ruthenium complex and FRET acceptor SYTO 61 is successfully exploited to amplify the luminescence in the presence of the mismatch. Luminescence and quenching studies with sodium iodide suggest that [Ru(bpy)2(dppz)](2+) binds to these mismatches via metalloinsertion from the minor groove. This work provides further evidence that metalloinsertion is a general binding mode of octahedral metal complexes to thermodynamically destabilized mismatches not only in DNA but also in RNA.

Tanshinones inhibit amyloid aggregation by amyloid-β peptide, disaggregate amyloid fibrils, and protect cultured cells.

The misfolding and aggregation of amyloid-β (Aβ) peptides into amyloid fibrils is regarded as one of the causative events in the pathogenesis of Alzheimer's disease (AD). Tanshinones extracted from Chinese herb Danshen (Salvia Miltiorrhiza Bunge) were traditionally used as anti-inflammation and cerebrovascular drugs due to their antioxidation and antiacetylcholinesterase effects. A number of studies have suggested that tanshinones could protect neuronal cells. In this work, we examine the inhibitory activity of tanshinone I (TS1) and tanshinone IIA (TS2), the two major components in the Danshen herb, on the aggregation and toxicity of Aβ1-42 using atomic force microscopy (AFM), thioflavin-T (ThT) fluorescence assay, cell viability assay, and molecular dynamics (MD) simulations. AFM and ThT results show that both TS1 and TS2 exhibit different inhibitory abilities to prevent unseeded amyloid fibril formation and to disaggregate preformed amyloid fibrils, in which TS1 shows better inhibitory potency than TS2. Live/dead assay further confirms that introduction of a very small amount of tanshinones enables protection of cultured SH-SY5Y cells against Aβ-induced cell toxicity. Comparative MD simulation results reveal a general tanshinone binding mode to prevent Aβ peptide association, showing that both TS1 and TS2 preferentially bind to a hydrophobic β-sheet groove formed by the C-terminal residues of I31-M35 and M35-V39 and several aromatic residues. Meanwhile, the differences in binding distribution, residues, sites, population, and affinity between TS1-Aβ and TS2-Aβ systems also interpret different inhibitory effects on Aβ aggregation as observed by in vitro experiments. More importantly, due to nonspecific binding mode of tanshinones, it is expected that tanshinones would have a general inhibitory efficacy of a wide range of amyloid peptides. These findings suggest that tanshinones, particularly TS1 compound, offer promising lead compounds with dual protective role in anti-inflammation and antiaggregation for further development of Aβ inhibitors to prevent and disaggregate amyloid formation.

HU Induced Perturbation to the Structure and Dynamics of Flexible DNA Substrate Construct

Many proteins modulate the structure and dynamics of the bacterial chromosome. One of the most abundant of such proteins is HU (Heat_Unstable) protein, a member of the DNABII family of DNA-binding proteins. An architectural protein, HU introduces sharp bends (120-160 degrees) into the DNA upon binding in a site-specific manner. DNA bending induced by these proteins is important for transcriptional regulation, initiation of replication, Mu transposition, base excision repair, recombination, and negative supercoiling in bacteria. HU exhibits a strong preference for various distortions in DNA, such as nicks, gaps, cruciforms, and sticky ends, all of which share a common motif of a flexible junction. There is little structural or dynamical information related to the perturbation of these DNA motifs upon HU binding. Characterization of HU binding to these motifs provides insight into HU's role in the regulation of DNA structure and also gives insight into the general mode of site-specific recognition and binding for non-sequence-specific DNA binding proteins found in both prokaryotes and eukaryotes. We have employed various fluorescence spectroscopic techniques to address this problem. Through the use of fluorescence anisotropy we have determined the dissociation constant in solution for HU binding to DNA overhang constructs to be 0.3 nM, which is a 1000-fold lower than binding to duplex DNA. The stoichiometry of HU binding specifically to these substrates is one-to-one, while non-specific binding generally leads to larger protein to DNA ratios in bound complexes. Forster resonance energy transfer (FRET) results are indicative of protein-induced bending of the DNA substrate at the duplex/single strand junction and in the single stand region upon HU binding. We further explore the mechanism of HU-induced bending, structural recognition and binding through the use of FRET mapping techniques and time-resolved fluorescence methods.

Computational modeling of human coreceptor CCR5 antagonist as a HIV-1 entry inhibitor: using an integrated homology modeling, docking, and membrane molecular dynamics simulation analysis approach

Chemokine receptor 5 (CCR5) is an integral membrane protein that is utilized during human immunodeficiency virus type-1 entry into host cells. CCR5 is a G-protein coupled receptor that contains seven transmembrane (TM) helices. However, the crystal structure of CCR5 has not been reported. A homology model of CCR5 was developed based on the recently reported CXCR4 structure as template. Automated docking of the most potent (14), medium potent (37), and least potent (25) CCR5 antagonists was performed using the CCR5 model. To characterize the mechanism responsible for the interactions between ligands (14, 25, and 37) and CCR5, membrane molecular dynamic (MD) simulations were performed. The position and orientation of ligands (14, 25, and 37) were found to be changed after MD simulations, which demonstrated the ability of this technique to identify binding modes. Furthermore, at the end of simulation, it was found that residues identified by docking were changed and some new residues were introduced in the proximity of ligands. Our results are in line with the majority of previous mutational reports. These results show that hydrophobicity is the determining factor of CCR5 antagonism. In addition, salt bridging and hydrogen bond contacts between ligands (14, 25, and 37) and CCR5 are also crucial for inhibitory activity. The residues newly identified by MD simulation are Ser160, Phe166, Ser180, His181, and Trp190, and so far no site-directed mutagenesis studies have been reported. To determine the contributions made by these residues, additional mutational studies are suggested. We propose a general binding mode for these derivatives based on the MD simulation results of higher (14), medium (37), and lower (25) potent inhibitors. Interestingly, we found some trend for these inhibitors such as, salt bridge interaction between basic nitrogen of ligand and acidic Glu283 seemed necessary for inhibitory activity. Also, two aromatic pockets (pocket I – TM1-3 and pocket II – TM3-6) were linked by the central polar region in TM7, and the simulated inhibitors show important interactions with the Trp86, Tyr89, Tyr108, Phe112, Ile198, Tyr251, Leu255, and Gln280 and Glu283 residues. These results shed light on the usage of MD simulation to identify more stable, optimal binding modes of the inhibitors.

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gα(i/o) during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gα(i)·GDP. The crystal structures of Gα(i)·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gα(i) interaction features when compared with the only high resolution structure known with GL/Gα(i) interaction between RGS14 and Gα(i1.) Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gα(i)·GDP. A highly conserved "double Arg finger" sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gα(i). Together with the sequence alignment, we suggest that the LGN GL/Gα(i) interaction represents a general binding mode between GL motifs and Gα(i). We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors.

Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105° bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an α helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.

Anti-Cooperative and Cooperative Protein-Protein Interactions between TetR Isoforms on Synthetic Enhancers

Protein-protein interactions play an important role in determining the regulatory output of cis regulatory regions. In this work, we revisit the regulatory output functions recorded for the synthetic enhancers that contain binding sites for TetR. We use our thermodynamic model as an analysis tool to infer that two different types of interactions may take place between the TetR molecules. First, a strong mutually exclusive anti-cooperative interaction precludes the synthetic enhancer from being occupied by more than one AT (the aTc bound TetR isoform) protein, and a second weak cooperative interaction exists between the aTc-free TetR isoform (T). Consequently, this work highlights the power of the synthetic enhancer approach as a tool for studying protein-protein interactions via an experimentally verifiable prediction for the general mode of binding of the TetR repressor.