RESUMO
Human sine oculis homeobox homolog (SIX) 1 contains a homeodomain (HD), which is important for binding to DNA. In this study, we carried out structural studies on the HD of human SIX1 using nuclear magnetic resonance (NMR) spectroscopy. Its secondary structures and dynamics in solution were explored. HD is well-structured in solution, and our study shows that it contains three α-helices. Dynamics study indicates that the N- and C-terminal residues of HD are flexible in solution. HD of human SIX1 exhibits molecular interactions with a short double-strand DNA sequence evidenced by the 1 H-15 N-heteronuclear single quantum correlation (HSQC) and 19 F-NMR experiments. Our current study provides structural information for HD of human SIX1. Further studies indicate that this construct can be utilized to study SIX1 and DNA interactions.
Assuntos
DNA , Proteínas de Homeodomínio , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Estrutura Secundária de ProteínaRESUMO
The Zika virus (ZIKV) genome encodes a polyprotein that can be post-translationally processed into functional viral proteins. The viral protease is indispensable in the maturation of viral proteins. The Zika protease comprises of two components crucial for catalysis. The N-terminal region of NS3 contains the catalytic triad and approximately 40 amino acids of NS2B are essential for folding and protease activity. NS2B is a membrane protein with transmembrane domains that are critical for the localization of NS3 to the membrane. In this study, we expressed and purified full-length NS2B from ZIKV in E. coli. Purified NS2B was then reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. It was found that compared to wild type NS2B, NS2B C11S mutation in LMPG exhibited dispersed cross peaks in the 1H15N-HSQC spectrum, thereby suggesting the feasibility for structural characterization using solution NMR spectroscopy.
Assuntos
Detergentes/química , Micelas , Ressonância Magnética Nuclear Biomolecular , Fosfatidilgliceróis/química , Proteínas não Estruturais Virais , Zika virus , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Zika virus/química , Zika virus/genéticaRESUMO
Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with the cell membrane and the C-terminal region consists of three potential transmembrane regions. The secondary structure of the intact NS4A is not known as the previous structural studies were carried out on its fragments. In this study, we purified the full-length NS4A of DENV serotype 4 into dodecylphosphocholine (DPC) micelles. Solution NMR studies reveal that NS4A contains six helices in DPC micelles. The N-terminal three helices are amphipathic and interact with the membrane. The C-terminal three helices are embedded in micelles. Our results suggest that NS4A contains three transmembrane helices. Our studies provide for the first time structural information of the intact NS4A of DENV and will be useful for further understanding its role in viral replication.
Assuntos
Proteínas de Membrana/química , Micelas , Fosforilcolina/análogos & derivados , Estrutura Secundária de Proteína , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosforilcolina/química , Fosforilcolina/metabolismo , Ligação Proteica , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Zika virus (ZIKV) protease is a two-component complex in which NS3 contains the catalytic triad and NS2B cofactor region is important for protease folding and activity. A protease construct-eZiPro without the transmembrane domains of NS2B was designed. Structural study on eZiPro reveals that the Thr-Gly-Lys-Arg (TGKR) sequence at the C-terminus of NS2B binds to the active site after cleavage. The bZiPro construct only contains NS2B cofactor region and the N-terminus of NS3 without any artificial linker or protease cleavage site, giving rise to an empty pocket accessible to substrate and inhibitor binding. Herein, we demonstrate that the TGKR sequence of NS2B in eZiPro is dynamic. Peptides from NS2B with various lengths exhibit different binding affinities to bZiPro. TGKR binding to the active site in eZiPro does not affect protease binding to small-molecule compounds. Our results suggest that eZiPro will also be useful for evaluating small-molecule protease inhibitors.
Assuntos
Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Infecção por Zika virus/virologia , Zika virus/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeos/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteínas não Estruturais Virais/química , Proteínas Virais/química , Zika virus/químicaRESUMO
Dengue virus is an important human pathogen affecting people especially in tropical and subtropical regions. Its genome encodes seven non-structural proteins that are important for viral assembly and replication. Dengue NS2B is a membrane protein containing four transmembrane helices and involved in protein-protein interactions. Its transmembrane helices are critical for location of NS2B on the cell membrane while one cytoplasmic region composed of approximately 40 amino acids serves as a cofactor of viral NS3 protease by forming a tight complex with the N-terminal region of NS3. Here, we report the backbone resonance assignments for a dengue NS2B construct referred to as mini-NS2B containing only the transmembrane regions without NS3 cofactor region in detergent micelles. Mini-NS2B exhibits well-dispersed cross-peaks in the 1H-15N-HSQC spectrum and contains four helices in solution. The available mini-NS2B and its assignment will be useful for determining the structure of NS2B and identifying small molecules binding to the transmembrane regions.
Assuntos
Dengue , Peptídeo Hidrolases , Humanos , Micelas , Detergentes/química , Ressonância Magnética Nuclear Biomolecular , Proteínas não Estruturais Virais/químicaRESUMO
Zika virus (ZIKV) NS2B-NS3 protease is a validated antiviral target as it is essential for maturation of viral proteins. However, its negatively charged active site hinders the development of orthosteric small-molecule inhibitors. Fragment-based drug discovery (FBDD) is a powerful tool to generate novel chemical starting points against difficult drug targets. In this study, we scre ened a fragment compound library against the Zika protease using a primary thermal shift assay and identified twenty-two fragments which (bind to and) stabilize the protease. We then determined the X-ray crystal structures of two hits from different classes, all of which bind to the S1 pocket of the protease. We confirmed that these two fragments bind to the protease without inducing significant conformational changes using solution NMR spectroscopy. These fragment scaffolds serve as the starting point for subsequent lead compound development.
Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Zika virus/efeitos dos fármacos , Antivirais/química , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Serina Endopeptidases , Zika virus/enzimologiaRESUMO
Zika virus (ZIKV) infection has become a global public health concern. The viral NS2B-NS3 protease is an attractive antiviral target because of its role in maturation of viral non-structural proteins. Substrate-derived protease inhibitors have been investigated, but it remains challenging to develop them into drugs. Small-molecule inhibitors are of great interest in antiviral drug development. Here we report the structure and dynamics of ZIKV NS2B-NS3 protease covalently bound to a small-molecule inhibitor. Our crystallographic and NMR studies demonstrate that the inhibitor further stabilizes the closed conformation of ZIKV protease. Upon hydrolysis in situ into two fragments, the benzoyl group of the inhibitor forms a covalent bond with the side chain of catalytic residue S135, whereas the second fragment exhibits no obvious molecular interactions with the protease. This study provides a detailed mechanism of action for a covalent inhibitor, which will guide further development of ZIKV protease inhibitors.
Assuntos
Antivirais/química , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Proteínas não Estruturais Virais/química , Zika virus/química , Sequência de Aminoácidos , Antivirais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Inibidores de Proteases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Zika virus/enzimologia , Zika virus/genéticaRESUMO
The Zika virus (ZIKV) NS2B-NS3 protease is an important drug target. The conventional flaviviral protease constructs used for structural studies contain the NS2B cofactor region linked to the NS3 protease domain via a glycine-rich flexible linker. Here, we examined the structural dynamics of this conventional Zika protease (gZiPro) using NMR spectroscopy. Although the glycine-rich linker in gZiPro does not alter the overall folding of the protease in solution, gZiPro is not homogenous in ion exchange chromatography. Compared to the unlinked protease construct, the artificial linker affects the chemical environment of many residues including H51 in the catalytic triad. Our study provides a direct comparison of ZIKV protease constructs with and without an artificial linker.
Assuntos
Proteínas Recombinantes/química , Serina Endopeptidases/química , Proteínas Virais/química , Zika virus/enzimologia , Domínio Catalítico , Cromatografia por Troca Iônica , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/genética , Serina Endopeptidases/genética , Inibidores de Serina Proteinase/química , Proteínas Virais/genéticaRESUMO
The NS2B-NS3 viral protease is an attractive drug target against Zika virus (ZIKV) due to its importance in viral replication and maturation. Here we report the crystal structure of protease in complex with a dipeptide inhibitor, Acyl-KR-aldehyde (compound 1). The aldehyde moiety forms a covalent bond with the catalytic Ser135 of NS3. The Arg and Lys residues in the inhibitor occupy the S1 and S2 sites of the protease, respectively. Nuclear magnetic resonance studies demonstrate that the complex is in the closed conformation in solution. The chemical environment of residues surrounding the active site is sensitive to the bound inhibitor as demonstrated by the comparison with two other non-covalent dipeptides, Acyl-K-Agmatine (compound 2) and Acyl-KR-COOH (compound 3). Removing the aldehyde moiety in 1 converts the binding mode from a slow to a fast exchange regime. The structural dynamics information obtained in this study will guide future drug discovery against ZIKV and other flaviviruses.
Assuntos
Antivirais/farmacologia , Dipeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/química , Antivirais/química , Domínio Catalítico , Dipeptídeos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Ligação Proteica , RNA Helicases/química , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Zika virus/enzimologiaRESUMO
Zika virus (ZIKV) has rapidly emerged as a global public health concern. Viral NS2B-NS3 protease processes viral polyprotein and is essential for the virus replication, making it an attractive antiviral drug target. We report crystal structures at 1.58-angstrom resolution of the unlinked NS2B-NS3 protease from ZIKV as free enzyme and bound to a peptide reversely oriented at the active site. The unlinked NS2B-NS3 protease adopts a closed conformation in which NS2B engages NS3 to form an empty substrate-binding site. A second protease in the same crystal binds to the residues K14K15G16E17 from the neighboring NS3 in reverse orientation, resisting proteolysis. These features of ZIKV NS2B-NS3 protease may accelerate the discovery of structure-based antiviral drugs against ZIKV and related pathogenic flaviviruses.
Assuntos
Proteínas não Estruturais Virais/química , Zika virus/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Oligopeptídeos/química , Ligação Proteica , Conformação Proteica , Proteólise , RNA Helicases/química , Serina Endopeptidases/química , Especificidade por SubstratoRESUMO
The recent outbreak of Zika virus (ZIKV) infections in the Americas represents a serious threat to the global public health. The viral protease that processes viral polyproteins during infection appears as an attractive drug target. Here we report a crystal structure at 1.84 Å resolution of ZIKV non-structural protein NS2B-NS3 protease with the last four amino acids of the NS2B cofactor bound at the NS3 active site. This structure represents a post-proteolysis state of the enzyme during viral polyprotein processing and provides insights into peptide substrate recognition by the protease. Nuclear magnetic resonance (NMR) studies and protease activity assays unravel the protein dynamics upon binding the protease inhibitor BPTI in solution and confirm this finding. The structural and functional insights of the ZIKV protease presented here should advance our current understanding of flavivirus replication and accelerate structure-based antiviral drug discovery against ZIKV.