Binding Cleft Research Articles

MD simulation reveals differential binding of Cm(III) and Th(IV) with serum transferrin at acidic pH.

The iron carrier human serum transferrin (sTf) is known to transport other metals, including some actinides (An). Radiotoxic An are routinely involved in the nuclear fuel cycle and the possibility of their accidental exposure cannot be ruled out. Understanding An interaction with sTf assumes a greater significance for the development of safe and efficacious chelators for their removal from the blood stream. Here we report several 100 ns equilibrium MD simulations of Cm(III)- and Th(IV)-loaded sTf at various protonation states of the protein to explore the possibility of the two An ions release and speciation. The results demonstrate variation in protonation state of dilysine pair (K206 and K296) and the tyrosine (Y188) residue is necessary for the opening of Cm(III)-bound protein and the release of the ion. For the tetravalent thorium, protonation of dilysine pair suffices to cause conformational changes of protein. However, in none of the protonation states, Th(IV) releases from sTf because of its strong electrostatic interaction with D63 in the first shell of the sTf binding cleft. Analysis of hydrogen bond, water bridge, and the evaluation of potential of mean forces of the An ions' release from sTf, substantiate the differential behavior of Cm(III) and Th(IV) at endosomal pH. The results provide insight in the regulation of Cm(III) and Th(IV) bioavailability that may prove useful for effective design of their decorporating agents and as well may help the future design of radiotherapy based on tetravalent ions.

Transient Unfolding and Long-Range Interactions in Viral BCL2 M11 Enable Binding to the BECN1 BH3 Domain.

Viral BCL2 proteins (vBCL2s) help to sustain chronic infection of host proteins to inhibit apoptosis and autophagy. However, details of conformational changes in vBCL2s that enable binding to BH3Ds remain unknown. Using all-atom, multiple microsecond-long molecular dynamic simulations (totaling 17 s) of the murine -herpesvirus 68 vBCL2 (M11), and statistical inference techniques, we show that regions of M11 transiently unfold and refold upon binding of the BH3D. Further, we show that this partial unfolding/refolding within M11 is mediated by a network of hydrophobic interactions, which includes residues that are 10 Å away from the BH3D binding cleft. We experimentally validate the role of these hydrophobic interactions by quantifying the impact of mutating these residues on binding to the Beclin1/BECN1 BH3D, demonstrating that these mutations adversely affect both protein stability and binding. To our knowledge, this is the first study detailing the binding-associated conformational changes and presence of long-range interactions within vBCL2s.

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2*.

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.

Crystal structure of SARS-CoV-2 papain-like protease

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC50 of 2.2 ± 0.3 μmol/L. We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3–S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.

Structural insights of the enzymes from the chitin utilization locus of Flavobacterium johnsoniae

Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (β-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.

Comparative structural analyses and nucleotide-binding characterization of the four KH domains of FUBP1

The FUBP1-FUSE complex is an essential component of a transcription molecular machinery that is necessary for tight regulation of expression of many key genes including c-Myc and p21. FUBP1 utilizes its four articulated KH modules, which function cooperatively, for FUSE nucleotide binding. To understand molecular mechanisms fundamental to the intermolecular interaction, we present a set of crystal structures, as well ssDNA-binding characterization of FUBP1 KH domains. All KH1-4 motifs were highly topologically conserved, and were able to interact with FUSE individually and independently. Nevertheless, differences in nucleotide binding properties among the four KH domains were evident, including higher nucleotide-binding potency for KH3 as well as diverse nucleotide sequence preferences. Variations in amino acid compositions at one side of the binding cleft responsible for nucleobase resulted in diverse shapes and electrostatic charge interaction, which might feasibly be a contributing factor for different nucleotide-binding propensities among KH1-4. Nonetheless, conservation of structure and nucleotide-binding property in all four KH motifs is essential for the cooperativity of multi KH modules present in FUBP1 towards nanomolar affinity for FUSE interaction. Comprehensive structural comparison and ssDNA binding characteristics of all four KH domains presented here provide molecular insights at a fundamental level that might be beneficial for elucidating the mechanisms of the FUBP1-FUSE interaction.

Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce β-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

Defective heart chamber growth and myofibrillogenesis after knockout of adprhl1 gene function by targeted disruption of the ancestral catalytic active site.

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3–4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

Engineering caspase 7 as an affinity reagent to capture proteolytic products.

Many proteases recognize their substrates with high specificities, with this in mind, it should theoretically be possible to utilize the substrate binding cleft of a protease as a scaffold to engineer an affinity reagent. In this study, we sought to develop reagents that would differentiate between substrates and products of proteolysis, based on a caspase 7 scaffold. Firstly, we engineered a form of caspase 7 that can undergo conversion to a substrate binding conformation without catalysis. Seeking to generate a product-only trap, we further engineered this construct by incorporating mutations that compensate for the generation of a negative charge in the neo C terminus of a newly generated product. This was accomplished with only three substitutions within the substrate binding cleft. Moreover, the affinity of the product trap for peptides was comparable to the affinity of caspase 7 to parental substrates. Finally, generation of a hybrid fluorescent protein with the product trap provided a reagent that specifically recognized apoptotic cells and highlights the versatility of such an approach in developing affinity and imaging agents for a variety of cysteine and serine proteases.

How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

AbstractExpanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4‐dioxane, dimethyl sulfoxide, 2,2,2‐trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H‐bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with “BSLA‐SSM” variant library (3439 variants; all natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments.

Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.

Soluble HLA-G expression levels and HLA-G/irinotecan association in metastatic colorectal cancer treated with irinotecan-based strategy

We here explore the soluble Human Leukocyte Antigen-G (sHLA-G) expression level as clinical biomarker in metastatic colorectal cancer (mCRC). To this aim the sHLA-G protein was measured in plasma samples of 40 patients with mCRC treated with the FOLFIRI (irinotecan (CPT-11) plus 5-fluorouracil (5-FU) and leucovorin (LV)) regimen. The results suggest a link between HLA-G levels and irinotecan (CPT-11) pharmacokinetic, leading to hypothesize a molecular interaction between sHLA-G and CPT-11. This interaction was confirmed experimentally by fluorescence spectroscopy. HLA-G is known to exist in a number of polymorphs that affect both the protein expression levels and its peptide-binding cleft. The interaction between HLA-G polymorphs and CPT-11 was explored by means of computational modelling, confirming the hypothesis that CPT-11 could actually target the peptide binding cleft of the most common HLA-G polymorphs.

In Silico Characterization of a Unique Plant-Like “Loopful” GH19 Chitinase from Newly Isolated Chitinophaga sp. YS-16

Chitinases are glycohydrolases that cleave β-1,4 linkages in chitin and are grouped into glycohydrolase (GH) family 18 and 19. GH18 chitinases are found ubiquitously, whereas GH19 chitinases are confined predominantly to higher plants and few bacteria. In this study, a new chitinolytic bacterium, Chitinophaga sp. YS-16, was isolated and identified as a close relative of Chitinophaga pinensis DSM 2588 with 99.41% similarity of 16S rRNA sequence. The gene encoding for GH19 chitinase bearing unique domain architecture was amplified from the genomic DNA of YS-16. The sequence of GH19 chitinase (CpChi) encoded in the genome of YS-16 showed 90.5% identity with the sequence of GH19 chitinase encoded in genome of C. pinensis DSM 2588 (ACU62980.1). Bioinformatics analyses of CpChi revealed it to be a 'loopful' chitinase which has been reported only from plants till date. The tertiary structure of the catalytic domain of CpChi (CtCpChi) was predicted by homology modelling. The docking of modelled structure of CtCpChi with oligomers of N-acetyl-D-glucosamine (GlcNAc) revealed that the binding cleft of CtCpChi could accommodate an octamer of GlcNAc, not known in any other reported bacterial chitinase. Phylogenetic analysis supports CtCpChi to be plant-like GH19 chitinase.

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery

Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high-resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C-terminal 320 residues adopt the fold of an eight-bladed β-propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N-terminal domain, which in Chlamydomonas ODA16 is located on top of the β-propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull-down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.

Target-Based Design of Promysalin Analogues Identifies a New Putative Binding Cleft in Succinate Dehydrogenase.

Promysalin is a small-molecule natural product that specifically inhibits growth of the Gram-negative pathogen Pseudomonas aeruginosa (PA). This activity holds promise in the treatment of multidrug resistant infections found in immunocompromised patients with chronic illnesses, such as cystic fibrosis. In 2015, our lab completed the first total synthesis; subsequent analogue design and SAR investigation enabled identification of succinate dehydrogenase (Sdh) as the biological target in PA. Herein, we report the target-guided design of new promysalin analogues with varying alkyl chains, one of which is on par with our most potent analogue to date. Computational docking revealed that some analogues have a different orientation in the Sdh binding pocket, placing the terminal carbon proximal to a tryptophan residue. This inspired the design of an extended side chain analogue bearing a terminal phenyl moiety, providing a basis for the design of future analogues.

Biophysical Folding Sensors for the Detection of Misfolded Proteins by Hsp70

Chaperoned ubiquitination complexes play a defining role in responding to chemical and pathological stresses and an understanding of how these complexes are regulated on a molecular level offers the opportunity to identify new avenues of treatment for a wide range of diseases. Hsp70 participates in chaperoned ubiquitination by recruiting and attempting to refold misfolded protein “clients” in an ATP‐dependent process. Misfolded clients interact with a binding cleft within the substrate binding domain (SBD) formed by a beta‐sheet subdomain (SBD‐beta) and helical lid subdomain. Understanding how Hsp70 recruits clients and positions clients relative to the E3 ligase CHIP is critical for understanding the core interactions between CHIP and Hsp70 that dictate the outcome of chaperoned ubiquitination. Herein we describe a set of folding sensors representing a spectrum of folded and unfolded states. When utilized in ubiquitination reactions this system provides a tunable folded/unfolded client with an Hsp70‐SBD binding site that provides control over the distance from the model unfolded client to the E2~Ub active site bound to the E3 ligase CHIP, thereby providing a measure of how folding state dictates degree of ubiquitination.Support or Funding InformationNIGMS R35 GM128595

Transcriptional Activation of Tumor Suppressor p73 is Modulated by MCL1 Through a Novel Protein Interaction

Precise regulation of apoptosis and cellular response to DNA damage are central to the development of multi‐cellular organisms. In eukaryotes, the BCL2 family of pro‐ and anti‐apoptotic proteins mediate multiple pro‐survival and stress signals to regulate mitochondrial membrane integrity and ultimately apoptosis. Likewise, the p53 family of transcription factors activate multiple pro‐death and cellular senescence pathways in response to DNA damage. Recent studies to develop anti‐cancer agents targeting the BCL2 family have demonstrated that one of the anti‐apoptotic proteins, MCL1, is a critical therapeutic target as it has been shown to inhibit apoptosis in response to a variety of chemotherapeutic agents. MCL1 activity centers around the availability of its BH3 binding cleft to form heterodimers with other pro‐apoptotic BCL2 family proteins. In our search for novel agents that could inhibit these interactions, we identified a novel peptide motif, that we call the reverse BH3 (rBH3) motif, that can interact with the BH3 cleft of MCL1. Here, we show that the p53 homolog, p73, contains a rBH3 motif in its tetramerization domain and that this motif mediates a direct protein‐protein interaction between MCL1 and p73. Furthermore, we show that this interaction allows MCL1 to regulate p73 target gene activation. With MCL1 inhibitors now in clinical trials, we are interrogating the impact that pharmacological inhibition of MCL1 has on DNA damage response signaling in cancer cells who rely on p73 for induction of cell cycle arrest and apoptosis.Support or Funding InformationNIH grant R01GM117391 to WJP

Ligand-induced conformational changes in a SMALP-encapsulated GPCR.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

Identification of functional domains of the minor fimbrial antigen involved in the interaction of Porphyromonas gingivalis with oral streptococci.

Porphyromonas gingivalis is associated with chronic periodontitis and may initially colonize the oral cavity by adhering to streptococci. Adhesion to streptococci is driven by interaction of the minor fimbrial antigen (Mfa1) with streptococcal antigen I/II. We identified the region of antigen I/II required for this interaction and developed small molecule mimetics that inhibited P. gingivalis adherence. However, the functional motifs of Mfa1 involved in the interaction with antigen I/II remain uncharacterized. A series of N‐ and C‐terminal peptide fragments of Mfa1 were expressed and tested for inhibition of P. gingivalis adherence to S. gordonii. This approach identified residues 225–400 of Mfa1 as essential for P. gingivalis adherence. Using the three‐dimensional structure of Mfa1, a putative binding cleft was identified using SiteMap and five small molecule mimetics could dock in this site. Site‐specific mutation of residues in the predicted cleft, including R240A, W275A, D321A and A357P inhibited the interaction of Mfa1 with streptococci, whereas mutation of residues not in the predicted cleft (V238A, I252F and ΔK253) had no effect. Complementation of an Mfa1‐deficient P. gingivalis strain with wild‐type mfa1 restored adherence to streptococci, whereas complementation with full‐length mfa1 containing the R240A or A357P mutations did not restore adherence. The mutations did not affect polymerization of Mfa1, suggesting that the complemented strains produced intact minor fimbriae. These results identified specific residues and structural motifs required for the Mfa1‐antigen I/II interaction and will facilitate the design of small molecule therapeutics to prevent P. gingivalis colonization of the oral cavity.

GPR6 Structural Insights: Homology Model Construction and Docking Studies.

GPR6 is an orphan G protein-coupled receptor that has been associated with the cannabinoid family because of its recognition of a sub-set of cannabinoid ligands. The high abundance of GPR6 in the central nervous system, along with high constitutive activity and a link to several neurodegenerative diseases make GPR6 a promising biological target. In fact, diverse research groups have demonstrated that GPR6 represents a possible target for the treatment of neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. Several patents have claimed the use of a wide range of pyrazine derivatives as GPR6 inverse agonists for the treatment of Parkinson’s disease symptoms and other dyskinesia syndromes. However, the full pharmacological importance of GPR6 has not yet been fully explored due to the lack of high potency, readily available ligands targeting GPR6. The long-term goal of the present study is to develop such ligands. In this paper, we describe our initial steps towards this goal. A human GPR6 homology model was constructed using a suite of computational techniques. This model permitted the identification of unique GPR6 structural features and the exploration of the GPR6 binding crevice. A subset of patented pyrazine analogs were docked in the resultant GPR6 inactive state model to validate the model, rationalize the structure-activity relationships from the reported patents and identify the key residues in the binding crevice for ligand recognition. We will take this structural knowledge into the next phase of GPR6 project, in which scaffold hopping will be used to design new GPR6 ligands.