Molecular dynamics simulations shows real-time lid opening in Hsp70 chaperone.

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.

Uncovering allostery and regulation in SORCIN through molecular dynamics simulations.

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.

Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation.

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.

Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques.

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.

Optimized filopodia formation requires myosin tail domain cooperation.

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.

Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1.

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.

Functionality of Redox-Active Cysteines Is Required for Restriction of Retroviral Replication by SAMHD1.

SAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of non-cycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations.

The small-molecule 3G11 inhibits HIV-1 reverse transcription.

The small-molecule 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (3G11) inhibits HIV-1 replication in the human T cell line MT-2. Here, we showed that 3G11 specifically and potently blocks HIV-1 infection. By contrast, 3G11 did not block other retroviruses such as HIV-2, simian immunodeficiency virus (SIVmac ), bovine immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, N-tropic murine leukemia virus, B-tropic murine leukemia virus, and Moloney murine leukemia virus. Analysis of DNA metabolism by real-time PCR revealed that 3G11 blocks the formation of HIV-1 late reverse transcripts during infection prior to the first-strand transfer step. In agreement, an in vitro assay revealed that 3G11 blocks the enzymatic activity of HIV-1 reverse transcriptase as strong as nevirapine. Docking of 3G11 to the HIV-1 reverse transcriptase enzyme suggested a direct interaction between residue L100 and 3G11. In agreement, an HIV-1 virus bearing the reverse transcriptase change L100I renders HIV-1 resistant to 3G11, which suggested that the reverse transcriptase enzyme is the viral determinant for HIV-1 sensitivity to 3G11. Although NMR experiments revealed that 3G11 binds to the HIV-1 capsid, functional experiments suggested that capsid is not the viral determinant for sensitivity to 3G11. Overall, we described a novel non-nucleoside reverse transcription inhibitor that blocks HIV-1 infection.

Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect.

SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.

Identification of Small-Molecule Inhibitors of the HuR/RNA Interaction Using a Fluorescence Polarization Screening Assay Followed by NMR Validation.

The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3' or 5' untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z'-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction.

Measurement and interpretation of 15N-1H residual dipolar couplings in larger proteins.

A decade ago, Dr. L.E. Kay and co-workers described an ingenious HNCO-based triple-resonance experiment from which several protein backbone RDCs can be measured simultaneously (Yang et al. (1999) [1]). They implemented a J-scaling technique in the (15)N dimension of the 3D experiment to obtain the NH RDCs. We have used this idea to carry out J-scaling in a 2D (15)N-(1)H-TROSY experiment and have found it to be an excellent method to obtain NH RDCs for larger proteins upto 70 kDa, far superior to commonly used HSQC in-phase/anti-phase and HSQC/TROSY comparisons. Here, this method, dubbed "RDC-TROSY" is discussed in detail and the limits of its utility are assessed by simulations. Prominent in the latter analysis is the evaluation of the effect of amide proton flips on the "RDC-TROSY" linewidths. The details of the technical and computational implementations of these methods for the determination of domain orientations in 45-60 kDa Hsp70 chaperone protein constructs are described.

Allostery in Hsp70 chaperones is transduced by subdomain rotations.

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's. It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD? Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.

Parameterization of peptide 13C carbonyl chemical shielding anisotropy in molecular dynamics simulations.

NMR chemical shielding anisotropy (CSA) relaxation is an important tool in the study of dynamical processes in proteins and nucleic acids in solution. Herein, we investigate how dynamical variations in local geometry affect the chemical shielding anisotropy relaxation of the carbonyl carbon nucleus, using the following protocol: 1) Using density functional theory, the carbonyl (13)C' CSA is computed for 103 conformations of the model peptide group N-methylacetamide (NMA). 2) The variations in computed (13)C' CSA parameters are fitted against quadratic hypersurfaces containing cross terms between the variables. 3) The predictive quality of the CSA hypersurfaces is validated by comparing the predicted and de novo calculated (13)C' CSAs for 20 molecular dynamics snapshots. 4) The CSA fluctuations and their autocorrelation and cross correlation functions due to bond-length and bond-angle distortions are predicted for a chemistry Harvard molecular mechanics (CHARMM) molecular dynamics trajectory of Ca(2+)-saturated calmodulin and GB3 from the hypersurfaces, as well as for a molecular dynamics (MD) simulation of an NMA trimer using a quantum mechanically correct forcefield. We find that the fluctuations can be represented by a 0.93 scaling factor of the CSA tensor for both R(1) and R(2) relaxations for residues in helix, coil, and sheet alike. This result is important, as it establishes that (13)C' relaxation is a valid tool for measurement of interesting dynamical events in proteins.