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1.
Eur Biophys J ; 52(4-5): 387-392, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37130969

RESUMO

Viral vector-based gene therapies and vaccines require accurate characterization of capsid species. The current gold standard for assessing capsid loading of adeno-associated virus (AAV) is sedimentation velocity analytical ultracentrifugation (SV-AUC). However, routine SV-AUC analysis is often size-limited, especially without the use of advanced techniques (e.g., gravitational-sweep) or when acquiring the multiwavelength data needed for assessing the loading fraction of viral vectors, and requires analysis by specialized software packages. Density gradient equilibrium AUC (DGE-AUC) is a highly simplified analytical method that provides high-resolution separation of biologics of different densities (e.g., empty and full viral capsids). The analysis required is significantly simpler than SV-AUC, and larger viral particles such as adenovirus (AdV) are amenable to characterization by DGE-AUC using cesium chloride gradients. This method provides high-resolution data with significantly less sample (estimated 56-fold improvement in sensitivity compared to SV-AUC). Multiwavelength analysis can also be used without compromising data quality. Finally, DGE-AUC is serotype-agnostic and amenable to intuitive interpretation and analysis (not requiring specialized AUC software). Here, we present suggestions for optimizing DGE-AUC methods and demonstrate a high-throughput AdV packaging analysis with the AUC, running as many as 21 samples in 80 min.


Assuntos
Ultracentrifugação , Ultracentrifugação/métodos
2.
Proc Natl Acad Sci U S A ; 116(44): 22196-22204, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31611382

RESUMO

Filopodia are actin-filled protrusions employed by cells to interact with their environment. Filopodia formation in Amoebozoa and Metazoa requires the phylogenetically diverse MyTH4-FERM (MF) myosins DdMyo7 and Myo10, respectively. While Myo10 is known to form antiparallel dimers, DdMyo7 lacks a coiled-coil domain in its proximal tail region, raising the question of how such divergent motors perform the same function. Here, it is shown that the DdMyo7 lever arm plays a role in both autoinhibition and function while the proximal tail region can mediate weak dimerization, and is proposed to be working in cooperation with the C-terminal MF domain to promote partner-mediated dimerization. Additionally, a forced dimer of the DdMyo7 motor is found to weakly rescue filopodia formation, further highlighting the importance of the C-terminal MF domain. Thus, weak dimerization activity of the DdMyo7 proximal tail allows for sensitive regulation of myosin activity to prevent inappropriate activation of filopodia formation. The results reveal that the principles of MF myosin-based filopodia formation are conserved via divergent mechanisms for dimerization.


Assuntos
Miosinas/metabolismo , Proteínas de Protozoários/metabolismo , Pseudópodes/metabolismo , Dictyostelium , Miosinas/química , Domínios Proteicos , Multimerização Proteica , Proteínas de Protozoários/química
3.
J Biol Chem ; 294(15): 6094-6112, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30808708

RESUMO

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.


Assuntos
Cálcio/química , Calmodulina/química , Canais de Potássio KCNQ/química , Animais , Células CHO , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cricetulus , Cristalografia por Raios X , Humanos , Ativação do Canal Iônico , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
4.
Eur Biophys J ; 49(8): 719-727, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32870347

RESUMO

Analytical ultracentrifugation (AUC) cells use either quartz or sapphire windows as end caps for the cell housing. Current generation sapphire windows are not recommended for absorbance data collection below 235 nm, because the window material shows a precipitous drop in transmittance at low wavelengths due to impurities in the sapphire. Quartz windows can be used below 235 nm as they do not exhibit adverse transmittance at low wavelengths. In this study, we demonstrate the optical properties of new generation sapphire windows and compare them to those of quartz windows across a wide range of wavelengths and present the results of sedimentation velocity experiments on BSA using both types of windows using data collected at both the 280 nm absorbance maxima as well as the 230-240 nm (closer to the peptide bond maximum). Our results show that the quartz and new generation sapphire windows deliver identical results in absorbance mode. We also demonstrate that quartz windows suffer significant mechanical deformation while spinning at very high speeds, while sapphire windows do not. This renders Rayleigh interference mode data collected at high speeds using quartz windows much noisier than with sapphire windows-which we have quantified by measuring how the signal to noise ratio of Fourier transformed Rayleigh interference scans degrades at high speed. Thus, we conclude that new-generation sapphire windows can be used for all AUC experiments through almost the entire mid UV range-obviating the need for quartz windows, unless wavelengths below 220 nm must be accessed.


Assuntos
Óxido de Alumínio , Quartzo , Ultracentrifugação/métodos , Animais , Controle de Qualidade , Soroalbumina Bovina/isolamento & purificação
5.
J Chem Inf Model ; 60(12): 6377-6391, 2020 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-33135886

RESUMO

The sterile α motif and histidine-aspartate domain-containing protein 1 (or SAMHD1) is a human protein that restricts HIV-1 in select terminally differentiated cells of the immune system by acting as a triphosphohydrolase, lowering dNTP pools. The functionally active form of the protein has been reported to be a tetramer where adjacent monomers are linked by GTP-Mg+2-dNTP cross-bridges, although some studies have also suggested the existence of a dimeric form of this protein. In this in silico study, we have investigated the stability of SAMHD1 dimeric "hold states" as well as the role of intrachain disulfide bonds. We have found that dimeric-GTP bound SAMHD1 can exist as a viable meso-stable hold state with extensive motion in the C-terminal domain, which is quenched upon tetramer assembly. The redox switch comprised of residues C341, C350, and C522 was found to be linked to changes in the allosteric site, suggesting a mechanism for initiating tetramer disassembly. The disulfide state of the protein dimer (C341-S-S-C350 vs C341-S-S-C522) also plays a role in driving affinities for the allosteric dATP molecules. In sum, our results suggest a model wherein dimeric SAMHD1 exists as a "hold state" in the cytosol, ready to be activated by dATP concentrations, where the "tunability" of this activation is further regulated by the redox state of the enzyme.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteína 1 com Domínio SAM e Domínio HD/química , Sítio Alostérico , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Oxirredução
6.
Proteins ; 87(9): 748-759, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31017331

RESUMO

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a "Scorched Earth" dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein-SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K m of ~10 µM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 µM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.


Assuntos
Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Oxirredução , Estrutura Secundária de Proteína , Proteína 1 com Domínio SAM e Domínio HD/genética
7.
J Virol ; 92(4)2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29187540

RESUMO

Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 µM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , HIV-1/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Restrição Antivirais , Aotidae , Proteínas do Capsídeo/ultraestrutura , Proteínas de Transporte/genética , HIV-1/metabolismo , Células HeLa , Humanos , Macaca mulatta , Domínios Proteicos , Multimerização Proteica , Proteínas/genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
8.
J Biol Chem ; 291(41): 21407-21413, 2016 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-27566548

RESUMO

SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC50dNTP values for SAMHD1 activation by dNTPs are in the 2-20 µm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism.


Assuntos
Desoxirribonucleotídeos/química , Proteínas Monoméricas de Ligação ao GTP/química , Mutação de Sentido Incorreto , Multimerização Proteica , Regulação Alostérica , Substituição de Aminoácidos , Desoxirribonucleotídeos/metabolismo , Ativação Enzimática , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Linfócitos T/enzimologia
9.
Proteins ; 85(7): 1266-1275, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28321930

RESUMO

The human sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a retroviral restriction factor in myeloid cells and non-cycling CD4+ T cells, a feature imputed to its phosphohydrolase activity-the enzyme depletes the cellular dNTP levels inhibiting reverse transcription. The functionally active form of SAMHD1 is an allosterically triggered tetramer which utilizes GTP-Mg+2 -dNTP cross bridges to link and stabilize adjacent monomers. However, very little is known about how it assembles into a tetramer and how long the tetramer stays intact. In this computational study, we provide a molecular dynamics based analysis of the structural stability and allosteric site dynamics in SAMHD1. We have investigated the allosteric links which assemble and hold the tetramer together. We have also extended this analysis to a regulatory mutant of SAMHD1. Experimental studies have indicated that phosphorylation of T592 downregulates HIV-1 restriction. A similar result is also achieved by a phosphomimetic mutation T592E. While a mechanistic understanding of the process is still elusive, the loss of structural integrity of the enzyme is conjectured to be the cause of the impaired dNTPase activity of the T592E mutant. MD simulations show that the T592E mutation causes slightly elevated local motions which remain confined to the short helix (residues 591-595), which contains the phosphorylation site and do not cause long-range destabilization of the SAMHD1 tetramer within the timeframe of the simulations. Thus, the regulatory mechanism of SAMHD1 is a more subtle mechanism than has been previously suspected. Proteins 2017; 85:1266-1275. © 2017 Wiley Periodicals, Inc.


Assuntos
Nucleotídeos de Desoxiadenina/química , Ácido Glutâmico/química , Guanosina Trifosfato/química , Proteínas Monoméricas de Ligação ao GTP/química , Treonina/química , Regulação Alostérica , Sítio Alostérico , Substituição de Aminoácidos , Nucleotídeos de Desoxiadenina/metabolismo , Ácido Glutâmico/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Magnésio , Simulação de Dinâmica Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Proteína 1 com Domínio SAM e Domínio HD , Especificidade por Substrato , Treonina/metabolismo
10.
J Chem Inf Model ; 57(10): 2523-2538, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-28956603

RESUMO

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. The catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP-triphosphohydrolase activity. The tetramer itself is assembled by a GTP/dNTP combination. This enzyme uses the strategy of deoxynucleotide starvation, which is thought to prevent effective reverse transcription of the retroviral genome-hence, restricting HIV-1 propagation. HIV-2 and SIV have evolved defenses against SAMHD1, underscoring its role in restriction. Previous studies have provided high-resolution structures of GTP/dNTP-bound enzyme complexes but have not been able to provide information on dynamics. In this study, we have used correlation network analysis along with MD techniques to study the flow of allosteric information across the active complex. We have found evidence of a reciprocal allosteric "handshake" occurring across monomeric units. We have also uncovered a short linker region as the nexus for funnelling the regulatory signal from phosphorylation at T592 from the surface to the interior core of the protein.


Assuntos
HIV-1/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Transdução de Sinais , Regulação Alostérica , HIV-1/química , Humanos , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/química , Especificidade por Substrato
11.
Proc Natl Acad Sci U S A ; 111(52): 18625-30, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25518861

RESUMO

Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.


Assuntos
Capsídeo/química , HIV-1/química , Indóis/química , Fenilalanina/análogos & derivados , Fatores de Poliadenilação e Clivagem de mRNA/química , Capsídeo/metabolismo , Cristalografia por Raios X , Infecções por HIV , HIV-1/metabolismo , Indóis/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/química , Fenilalanina/farmacologia , Ligação Proteica , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
12.
J Am Chem Soc ; 137(40): 12954-65, 2015 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-26306428

RESUMO

Approaches that combine experimental data and computational molecular dynamics (MD) to determine atomic resolution ensembles of biomolecules require the measurement of abundant experimental data. NMR residual dipolar couplings (RDCs) carry rich dynamics information, however, difficulties in modulating overall alignment of nucleic acids have limited the ability to fully extract this information. We present a strategy for modulating RNA alignment that is based on introducing variable dynamic kinks in terminal helices. With this strategy, we measured seven sets of RDCs in a cUUCGg apical loop and used this rich data set to test the accuracy of an 0.8 µs MD simulation computed using the Amber ff10 force field as well as to determine an atomic resolution ensemble. The MD-generated ensemble quantitatively reproduces the measured RDCs, but selection of a sub-ensemble was required to satisfy the RDCs within error. The largest discrepancies between the RDC-selected and MD-generated ensembles are observed for the most flexible loop residues and backbone angles connecting the loop to the helix, with the RDC-selected ensemble resulting in more uniform dynamics. Comparison of the RDC-selected ensemble with NMR spin relaxation data suggests that the dynamics occurs on the ps-ns time scales as verified by measurements of R(1ρ) relaxation-dispersion data. The RDC-satisfying ensemble samples many conformations adopted by the hairpin in crystal structures indicating that intrinsic plasticity may play important roles in conformational adaptation. The approach presented here can be applied to test nucleic acid force fields and to characterize dynamics in diverse RNA motifs at atomic resolution.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química , Conformação de Ácido Nucleico
13.
Proc Natl Acad Sci U S A ; 108(47): 18966-71, 2011 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-22065753

RESUMO

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70-DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1-70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual "tethered" binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422-433].


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Conformação Proteica , Dobramento de Proteína , Espectroscopia de Ressonância Magnética , Mutagênese , Ligação Proteica
14.
J Mol Graph Model ; 129: 108748, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38452417

RESUMO

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. While the prevailing hypothesis is that the catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP - triphosphohydrolase activity, it is also known to bind to ssRNA and ssDNA oligomers. A complete picture of the structure-function relationship of the enzyme is still elusive and the function corresponding to its nucleic acid binding ability is debated. In this in silico study, we investigate the stability, preference and allosteric effects of DNA oligomers bound to SAMHD1. In particular, we compare the binding of DNA and RNA oligomers of the same sequence and also consider the binding of DNA fragments with phosphorothioate bonds in the backbone. The results are compared with the canonical form with the monomers connected by GTP/dATP crossbridges. The simulations indicate that SAMHD1 dimers preferably bind to DNA and RNA oligomers compared to GTP/dATP. However, allosteric communication channels are altered in the nucleic acid acid bound complexes compared to the canonical form. All results are consistent with the hypothesis that the DNA bound form of the protein correspond to an unproductive off-pathway state where the protein is sequestered and not available for dNTP hydrolysis.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Monoméricas de Ligação ao GTP , Humanos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Nucleotídeos/metabolismo , DNA , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Comunicação , RNA
15.
J Biomol Struct Dyn ; 42(4): 1812-1825, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37098805

RESUMO

Soluble resistance-related calcium-binding protein or Sorcin is an allosteric, calcium-binding Penta-EF hand (PEF) family protein implicated in multi-drug resistant cancers. Sorcin is known to bind chemotherapeutic molecules such as Doxorubicin. This study uses in-silico molecular dynamics simulations to explore the dynamics and allosteric behavior of Sorcin in the context of Ca2+ uptake and Doxorubicin binding. The results show that Ca2+ binding induces large, but reversible conformational changes in the Sorcin structure which manifest as rigid body reorientations that preserve the local secondary structure. A reciprocal allosteric handshake centered around the EF5 hand is found to be key in Sorcin dimer formation and stabilization. Binding of Doxorubicin results in rearrangement of allosteric communities which disrupts long-range allosteric information transfer from the N-terminal domain to the middle lobe. However, this binding does not result in secondary structure destabilization. Sorcin does not appear to have a distinct Ca2+ activated mode of Doxorubicin binding.Communicated by Ramaswamy H. Sarma.


Assuntos
Simulação de Dinâmica Molecular , Neoplasias , Humanos , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Estrutura Secundária de Proteína , Neoplasias/tratamento farmacológico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Cálcio/química
16.
J Mol Graph Model ; 129: 108726, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38377794

RESUMO

The stress-inducible mammalian heat shock protein Hsp70 and its bacterial orthologue DnaK are highly conserved molecular chaperones and a crucial part of the machinery responsible for protein folding and homeostasis. Hsp70 is a three-domain, 70 kDa protein that cycles between an ATP-bound state in which all three domains are securely coupled into one unit and an ADP-bound state in which they are loosely attached via a flexible interdomain linker. The Hsp70 presents an alluring novel therapeutic target since it is crucial for maintaining cellular proteostasis and is particularly crucial to cancer cells. We have performed molecular dynamics simulations of the SBD (substrate binding domain) along with the Lid domain in response to experimental efforts to identify small molecule inhibitors that impair the functioning of Hsp70. Our intent has been to characterize the motion of the SBD/Lid allosteric machinery and in, addition, to identify the effect of the PET16 molecule on this motion. Interestingly, we noticed the opening of the entire Lid domain in the apo-form of the dimer. The configuration of the open structure was very different from previously published structures (PDB 4JN4) of the open and docked conformation of the ATP bound form. MD simulations revealed the Lid to be capable of far greater dynamical excursions than has been anticipated by experimental structural biology. This is of value in future drug discovery efforts targeted to modulating Hsp70 activity. The PET16 molecule appears to be weakly bound and its effect on the dynamics of the complex is yet to be elucidated.


Assuntos
Proteínas de Escherichia coli , Simulação de Dinâmica Molecular , Animais , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares , Trifosfato de Adenosina/metabolismo , Mamíferos/metabolismo
17.
Retrovirology ; 10: 131, 2013 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-24219908

RESUMO

BACKGROUND: SAMHD1 is a restriction factor that potently blocks infection by HIV-1 and other retroviruses. We have previously demonstrated that SAMHD1 oligomerizes in mammalian cells by immunoprecipitation. Here we investigated the contribution of SAMHD1 oligomerization to retroviral restriction. RESULTS: Structural analysis of SAMHD1 and homologous HD domain proteins revealed that key hydrophobic residues Y146, Y154, L428 and Y432 stabilize the extensive dimer interface observed in the SAMHD1 crystal structure. Full-length SAMHD1 variants Y146S/Y154S and L428S/Y432S lost their ability to oligomerize tested by immunoprecipitation in mammalian cells. In agreement with these observations, the Y146S/Y154S variant of a bacterial construct expressing the HD domain of human SAMHD1 (residues 109-626) disrupted the dGTP-dependent tetramerization of SAMHD1 in vitro. Tetramerization-defective variants of the full-length SAMHD1 immunoprecipitated from mammalian cells and of the bacterially-expressed HD domain construct lost their dNTPase activity. The nuclease activity of the HD domain construct was not perturbed by the Y146S/Y154S mutations. Remarkably, oligomerization-deficient SAMHD1 variants potently restricted HIV-1 infection. CONCLUSIONS: These results suggested that SAMHD1 oligomerization is not required for the ability of the protein to block HIV-1 infection.


Assuntos
HIV-1/imunologia , Interações Hospedeiro-Patógeno , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Multimerização Proteica , Linhagem Celular , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Imunoprecipitação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteína 1 com Domínio SAM e Domínio HD
18.
J Biomol Struct Dyn ; : 1-19, 2023 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-38063068

RESUMO

The Hsp70 chaperone protein system is an essential component of the protein folding and homeostasis machinery in E.Coli. Hsp70 is a three domain, 70 kDa protein which functions as an allosteric system cycling between an ADP-bound state where the three domains are loosely coupled via a flexible interdomain linker and an ATP-bound state where they are tightly coupled into a single entity. The structure-function model of this protein proposes an allosteric connection between the 45 kDa Nucleotide Binding Domain (NBD) and the 25 kDa Substrate Binding Domain (SBD) and Lid Domain which operates through the inter NBD-SBD linker. X-Ray crystallography and NMR spectroscopy have provided structures of the end states of the functional cycle of this protein, bound to ADP and ATP. We have used MD simulations to study the transitions between these end states and allosteric communication in this system. Our results largely validate the experimentally derived allosteric model of function, but shed additional light on the flow of allosteric information in the SBD + Lid. Specifically, we find that the Lid domain has a double-hinged structure with the potential for greater conformational flexibility than was hitherto expected.Communicated by Ramaswamy H. Sarma.

19.
Eur J Pharm Biopharm ; 189: 68-83, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37196871

RESUMO

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.


Assuntos
Capsídeo , Dependovirus , Dependovirus/genética , Dependovirus/química , Vetores Genéticos , Proteínas do Capsídeo , Ultracentrifugação , DNA
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