RESUMEN
Ubiquilins are a family of cytosolic proteins that ferry ubiquitinated substrates to the proteasome for degradation. Recent work has demonstrated that Ubiquilins can also act as molecular chaperones, utilizing internal Sti1 domains to directly bind to hydrophobic sequences. Ubiquilins are associated with several neurodegenerative diseases with point mutations in UBQLN2 causing dominant, X-linked Amyotrophic Lateral Sclerosis (ALS). The molecular basis of Ubiquilin chaperone activity and how ALS mutations in the Sti1 domains affect Ubiquilin activity are poorly understood. This study presents the first crystal structure of the Sti1 domain from a fungal Ubiquilin homolog bound to a transmembrane domain (TMD). The structure reveals that two Sti1 domains form a head-to-head dimer, creating a hydrophobic cavity that accommodates two TMDs. Mapping the UBQLN2 sequence onto the structure shows that several ALS mutations are predicted to disrupt the hydrophobic groove. Using a newly developed competitive binding assay, we show that Ubiquilins preferentially bind to hydrophobic substrates with low helical propensity, motifs that are enriched in both substrates and in Ubiquilins. This study provides insights into the molecular and structural basis for Ubiquilin substrate binding, with broad implications for the role of the Sti1 domain in phase separation and ALS.
RESUMEN
Transmembrane ß-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane ß-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.
Asunto(s)
Nanoporos , Ingeniería de Proteínas , Pliegue de Proteína , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Lámina beta , Modelos MolecularesRESUMEN
The main protease (MPro) of SARS-CoV-2 is crucial for the virus's replication and pathogenicity. Its active site is characterized by four distinct pockets (S1, S2, S4, and S1-3') and a solvent-exposed S3 site for accommodating a protein substrate. During X-ray crystallographic analyses of MPro bound with dipeptide inhibitors containing a flexible N-terminal group, we often observed an unexpected binding mode. Contrary to the anticipated engagement with the deeper S4 pocket, the N-terminal group frequently assumed a twisted conformation, positioning it for interactions with the S3 site and the inhibitor component bound at the S1 pocket. Capitalizing on this observation, we engineered novel inhibitors to engage both S3 and S4 sites or to adopt a rigid conformation for selective S3 site binding. Several new inhibitors demonstrated high efficacy in MPro inhibition. Our findings underscore the importance of the S3 site's unique interactions in the design of future MPro inhibitors as potential COVID-19 therapeutics.
RESUMEN
Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.
Asunto(s)
Clorofila , Clorofila/química , Clorofila/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Fotosíntesis , Transferencia de Energía , Microscopía por Crioelectrón , Conformación Proteica , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismoRESUMEN
Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.
Asunto(s)
Anticoncepción , Anticonceptivos Masculinos , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Bibliotecas de Moléculas Pequeñas , Animales , Humanos , Masculino , Ratones , Barrera Hematotesticular/metabolismo , Anticonceptivos Masculinos/química , Anticonceptivos Masculinos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Testículo/efectos de los fármacos , Anticoncepción/métodos , Relación Estructura-ActividadRESUMEN
N-myristoyltransferase (NMT) is a promising antimalarial drug target. Despite biochemical similarities between Plasmodium vivax and human NMTs, our recent research demonstrated that high selectivity is achievable. Herein, we report PvNMT-inhibiting compounds aimed at identifying novel mechanisms of selectivity. Various functional groups are appended to a pyrazole moiety in the inhibitor to target a pocket formed beneath the peptide binding cleft. The inhibitor core group polarity, lipophilicity, and size are also varied to probe the water structure near a channel. Selectivity index values range from 0.8 to 125.3. Cocrystal structures of two selective compounds, determined at 1.97 and 2.43 Å, show that extensions bind the targeted pocket but with different stabilities. A bulky naphthalene moiety introduced into the core binds next to instead of displacing protein-bound waters, causing a shift in the inhibitor position and expanding the binding site. Our structure-activity data provide a conceptual foundation for guiding future inhibitor optimizations.
Asunto(s)
Aciltransferasas , Antimaláricos , Inhibidores Enzimáticos , Plasmodium vivax , Pirazoles , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química , Plasmodium vivax/enzimología , Plasmodium vivax/efectos de los fármacos , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Aciltransferasas/química , Relación Estructura-Actividad , Antimaláricos/química , Antimaláricos/farmacología , Antimaláricos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Sitios de UniónRESUMEN
A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.
Asunto(s)
Nanoestructuras , Proteínas , Cristalografía por Rayos X , Nanoestructuras/química , Proteínas/química , Proteínas/metabolismo , Microscopía Electrónica , Reproducibilidad de los ResultadosRESUMEN
Cooperative interactions between amino acids are critical for protein function. A genetic reflection of cooperativity is epistasis, which is when a change in the amino acid at one position changes the sequence requirements at another position. To assess epistasis within an enzyme active site, we utilized CTX-M ß-lactamase as a model system. CTX-M hydrolyzes ß-lactam antibiotics to provide antibiotic resistance, allowing a simple functional selection for rapid sorting of modified enzymes. We created all pairwise mutations across 17 active site positions in the ß-lactamase enzyme and quantitated the function of variants against two ß-lactam antibiotics using next-generation sequencing. Context-dependent sequence requirements were determined by comparing the antibiotic resistance function of double mutations across the CTX-M active site to their predicted function based on the constituent single mutations, revealing both positive epistasis (synergistic interactions) and negative epistasis (antagonistic interactions) between amino acid substitutions. The resulting trends demonstrate that positive epistasis is present throughout the active site, that epistasis between residues is mediated through substrate interactions, and that residues more tolerant to substitutions serve as generic compensators which are responsible for many cases of positive epistasis. Additionally, we show that a key catalytic residue (Glu166) is amenable to compensatory mutations, and we characterize one such double mutant (E166Y/N170G) that acts by an altered catalytic mechanism. These findings shed light on the unique biochemical factors that drive epistasis within an enzyme active site and will inform enzyme engineering efforts by bridging the gap between amino acid sequence and catalytic function.
Asunto(s)
Escherichia coli , beta-Lactamasas , Escherichia coli/genética , Dominio Catalítico/genética , Mutación , Sustitución de Aminoácidos , beta-Lactamasas/químicaRESUMEN
The design of protein-protein interfaces using physics-based design methods such as Rosetta requires substantial computational resources and manual refinement by expert structural biologists. Deep learning methods promise to simplify protein-protein interface design and enable its application to a wide variety of problems by researchers from various scientific disciplines. Here, we test the ability of a deep learning method for protein sequence design, ProteinMPNN, to design two-component tetrahedral protein nanomaterials and benchmark its performance against Rosetta. ProteinMPNN had a similar success rate to Rosetta, yielding 13 new experimentally confirmed assemblies, but required orders of magnitude less computation and no manual refinement. The interfaces designed by ProteinMPNN were substantially more polar than those designed by Rosetta, which facilitated in vitro assembly of the designed nanomaterials from independently purified components. Crystal structures of several of the assemblies confirmed the accuracy of the design method at high resolution. Our results showcase the potential of deep learning-based methods to unlock the widespread application of designed protein-protein interfaces and self-assembling protein nanomaterials in biotechnology.
Asunto(s)
Nanoestructuras , Proteínas , Modelos Moleculares , Proteínas/química , Secuencia de Aminoácidos , Biotecnología , Conformación ProteicaRESUMEN
The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.
Asunto(s)
Orthoreovirus , Reoviridae , Animales , Orthoreovirus/genética , Replicación Viral , Reoviridae/genética , ARN/metabolismo , Ácidos y Sales Biliares , ARN Viral/genética , Mamíferos/genéticaRESUMEN
Natural proteins are highly optimized for function but are often difficult to produce at a scale suitable for biotechnological applications due to poor expression in heterologous systems, limited solubility, and sensitivity to temperature. Thus, a general method that improves the physical properties of native proteins while maintaining function could have wide utility for protein-based technologies. Here, we show that the deep neural network ProteinMPNN, together with evolutionary and structural information, provides a route to increasing protein expression, stability, and function. For both myoglobin and tobacco etch virus (TEV) protease, we generated designs with improved expression, elevated melting temperatures, and improved function. For TEV protease, we identified multiple designs with improved catalytic activity as compared to the parent sequence and previously reported TEV variants. Our approach should be broadly useful for improving the expression, stability, and function of biotechnologically important proteins.
Asunto(s)
Endopeptidasas , Temperatura , Endopeptidasas/metabolismo , Proteínas Recombinantes de FusiónRESUMEN
Vital to the treatment of influenza is the use of antivirals such as Oseltamivir (Tamiflu) and Zanamivir (Relenza); however, antiviral resistance is becoming an increasing problem for these therapeutics. The RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, a critical component of influenza viral replication machinery, is an antiviral target that was recently validated with the approval of Baloxavir Marboxil (BXM). Despite its clinical success, BXM has demonstrated susceptibility to resistance mutations, specifically the I38T, E23K, and A36 V mutants of PAN. To better understand the effects of these mutations on BXM resistance and improve the design of more robust therapeutics, this study examines key differences in protein-inhibitor interactions with two inhibitors and the I38T, E23K, and A36 V mutants. Differences in inhibitor binding were evaluated by measuring changes in binding to PAN using two biophysical methods. The binding mode of two distinct inhibitors was determined crystallographically with both wild-type and mutant forms of PAN. Collectively, these studies give some insight into the mechanism of antiviral resistance of these mutants.
Asunto(s)
Dibenzotiepinas , Gripe Humana , Morfolinas , Tiepinas , Humanos , Oxazinas , Piridinas/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Endonucleasas/genética , Tiepinas/farmacología , Tiepinas/uso terapéutico , Piridonas/uso terapéutico , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Zanamivir/uso terapéutico , Triazinas/farmacología , Triazinas/uso terapéuticoRESUMEN
Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.
Asunto(s)
Neoplasias de la Próstata , Masculino , Humanos , Ratones , Animales , Peso Molecular , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Tirosina , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
Klebsiella pneumoniae carbapenemase 2 (KPC-2) is an important source of drug resistance as it can hydrolyze and inactivate virtually all ß-lactam antibiotics. KPC-2 is potently inhibited by avibactam via formation of a reversible carbamyl linkage of the inhibitor with the catalytic serine of the enzyme. However, the use of avibactam in combination with ceftazidime (CAZ-AVI) has led to the emergence of CAZ-AVI-resistant variants of KPC-2 in clinical settings. One such variant, KPC-44, bears a 15 amino acid duplication in one of the active-site loops (270-loop). Here, we show that the KPC-44 variant exhibits higher catalytic efficiency in hydrolyzing ceftazidime, lower efficiency toward imipenem and meropenem, and a similar efficiency in hydrolyzing ampicillin, than the WT KPC-2 enzyme. In addition, the KPC-44 variant enzyme exhibits 12-fold lower AVI carbamylation efficiency than the KPC-2 enzyme. An X-ray crystal structure of KPC-44 showed that the 15 amino acid duplication results in an extended and partially disordered 270-loop and also changes the conformation of the adjacent 240-loop, which in turn has altered interactions with the active-site omega loop. Furthermore, a structure of KPC-44 with avibactam revealed that formation of the covalent complex results in further disorder in the 270-loop, suggesting that rearrangement of the 270-loop of KPC-44 facilitates AVI carbamylation. These results suggest that the duplication of 15 amino acids in the KPC-44 enzyme leads to resistance to CAZ-AVI by modulating the stability and conformation of the 270-, 240-, and omega-loops.
Asunto(s)
Ceftazidima , Farmacorresistencia Bacteriana , Modelos Moleculares , Humanos , Aminoácidos/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Ceftazidima/farmacología , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Farmacorresistencia Bacteriana/genética , Cristalografía por Rayos X , Dominio Catalítico/genética , Estructura Terciaria de ProteínaRESUMEN
ß-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important ß-lactam class of antibiotics. The OXA-48 and NDM-1 ß-lactamases cause resistance to the last-resort ß-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel ß-lactamase inhibitors. We exploited the ß-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all ß-lactamases. A library of 106 compounds, each containing a carboxylic acid or a tetrazole as an enzyme recognition element, was designed, constructed, and used to identify OXA-48 and NDM-1 inhibitors with micromolar to nanomolar potency. Further optimization led to NDM-1 inhibitors with increased potencies and biological activities. This work demonstrates that the carboxylate-binding pocket-targeting DECL, designed based on substrate binding information, aids in inhibitor identification and led to the discovery of novel non-ß-lactam pharmacophores for the development of ß-lactamase inhibitors for enzymes of different structural and mechanistic classes.
Asunto(s)
Antibacterianos , Inhibidores de beta-Lactamasas , Inhibidores de beta-Lactamasas/farmacología , Inhibidores de beta-Lactamasas/química , Antibacterianos/farmacología , Antibacterianos/química , beta-Lactamasas/metabolismo , beta-Lactamas/farmacología , Penicilinas , ADN , Pruebas de Sensibilidad MicrobianaRESUMEN
Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a "raised" conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.
Asunto(s)
Antígenos de Grupos Sanguíneos , Infecciones por Caliciviridae , Norovirus , Humanos , Proteínas de la Cápside/química , Cápside/metabolismo , Norovirus/genética , Sitios de Unión , Epítopos/metabolismo , Antígenos de Grupos Sanguíneos/metabolismoRESUMEN
Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain-side-chain interactions across protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein-protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.
Asunto(s)
Proteínas , Proteínas/química , CristalizaciónRESUMEN
Drugs targeting multiple stages of the Plasmodium vivax life cycle are needed to reduce the health and economic burdens caused by malaria worldwide. N-myristoyltransferase (NMT) is an essential eukaryotic enzyme and a validated drug target for combating malaria. However, previous PvNMT inhibitors have failed due to their low selectivity over human NMTs. Herein, we apply a structure-guided hybridization approach combining chemical moieties of previously reported NMT inhibitors to develop the next generation of PvNMT inhibitors. A high-resolution crystal structure of PvNMT bound to a representative selective hybrid compound reveals a unique binding site architecture that includes a selective conformation of a key tyrosine residue. The hybridized compounds significantly decrease P. falciparum blood-stage parasite load and consistently exhibit dose-dependent inhibition of P. vivax liver stage schizonts and hypnozoites. Our data demonstrate that hybridized NMT inhibitors can be multistage antimalarials, targeting dormant and developing forms of liver and blood stage.
Asunto(s)
Malaria Falciparum , Malaria Vivax , Humanos , Animales , Plasmodium vivax , Esquizontes , Hígado , AciltransferasasRESUMEN
Each year, approximately 50,000 children under 5 die as a result of diarrhea caused by Cryptosporidium parvum, a protozoan parasite. There are currently no effective drugs or vaccines available to cure or prevent Cryptosporidium infection, and there are limited tools for identifying and validating targets for drug or vaccine development. We previously reported a high throughput screening (HTS) of a large compound library against Plasmodium N-myristoyltransferase (NMT), a validated drug target in multiple protozoan parasite species. To identify molecules that could be effective against Cryptosporidium, we counter-screened hits from the Plasmodium NMT HTS against Cryptosporidium NMT. We identified two potential hit compounds and validated them against CpNMT to determine if NMT might be an attractive drug target also for Cryptosporidium. We tested the compounds against Cryptosporidium using both cell-based and NMT enzymatic assays. We then determined the crystal structure of CpNMT bound to Myristoyl-Coenzyme A (MyrCoA) and structures of ternary complexes with MyrCoA and the hit compounds to identify the ligand binding modes. The binding site architectures display different conformational states in the presence of the two inhibitors and provide a basis for rational design of selective inhibitors.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Plasmodium , Niño , Humanos , Criptosporidiosis/tratamiento farmacológico , Desarrollo de MedicamentosRESUMEN
Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha-helical coiled coils have such symmetric, extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibres can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibres can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.