Akash Bhattacharya

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.

Significance: Cells interact with their environment using filopodia, thin membrane protrusions supported by actin filaments. Filopodia formation in evolutionarily divergent organisms requires force generation by a MyTH4-FERM (MF) myosin that binds membrane proteins and is designed to promote cytoskeletal reorganization and filopodial transport. The amoeboid filopodial MF myosin is found to be a resilient motor in filopodia formation. Its activity and regulation appear to have emerged by multidomain coevolution for optimized control of filopodia formation and stability. Its properties reflect those of the ancestral MF myosin that gave rise to the metazoan MF myosins. These findings have implications for understanding the fundamental principles of how filopodia form and how MF myosins function in phylogenetically distant organisms.

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.

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 ( K _D 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 ( K _D 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.

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.