Joshua L. Phillips

A bimodal distribution of two distinct categories of intrinsically-disordered structures with separate functions in FG nucleoporins

Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties.

Plasma IgG to Linear Epitopes in the V2 and V3 Regions of HIV-1 gp120 Correlate with a Reduced Risk of Infection in the RV144 Vaccine Efficacy Trial

Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.

A biologically inspired working memory framework for robots

The human brain includes a capacity-limited memory system devoted to the short-term retention of task-relevant information. This system is called working memory. Some computational neuroscience accounts of working memory have explained it in terms of interactions between the prefrontal cortex and the mesolimbic dopamine system. Inspired by these models, we have constructed a software toolkit for creating working memory components for robot control systems, based on the proposed mechanisms used by the brain. We report our design for this toolkit, as well as the results of a feasibility study, involving a robotic version of the delayed saccade task, and we discuss future plans to test this framework in the context of more complex tasks.

A data-driven approach to modeling the tripartite structure of multidrug resistance efflux pumps.

Many bacterial pathogens are becoming increasingly resistant to antibiotic treatments, and a detailed understanding of the molecular basis of antibiotic resistance is critical for the development of next‐generation approaches for combating bacterial infections. Studies focusing on pathogens have revealed the profile of resistance in these organisms to be due primarily to the presence of multidrug resistance efflux pumps: tripartite protein complexes which span the periplasm bridging the inner and outer membranes of Gram‐negative bacteria. An atomic‐level resolution tripartite structure remains imperative to advancing our understanding of the molecular mechanisms of pump function using both theoretical and experimental approaches. We develop a fast and consistent method for constructing tripartite structures which leverages existing data‐driven models and provide molecular modeling approaches for constructing tripartite structures of multidrug resistance efflux pumps. Our modeling studies reveal that conformational changes in the inner membrane component responsible for drug translocation have limited impact on the conformations of the other pump components, and that two distinct models derived from conflicting experimental data are both consistent with all currently available measurements. Additionally, we investigate putative drug translocation pathways via geometric simulations based on the available crystal structures of the inner membrane pump component, AcrB, bound to two drugs which occupy distinct binding sites: doxorubicin and linezolid. These simulations suggest that smaller drugs may enter the pump through a channel from the cytoplasmic leaflet of the inner membrane, while both smaller and larger drug molecules may enter through a vestibule accessible from the periplasm. Proteins 2015; 83:46–65.

Dynamic electrophoretic fingerprinting of the HIV-1 envelope glycoprotein

BackgroundInteractions between the HIV-1 envelope glycoprotein (Env) and its primary receptor CD4 are influenced by the physiological setting in which these events take place. In this study, we explored the surface chemistry of HIV-1 Env constructs at a range of pH and salinities relevant to mucosal and systemic compartments through electrophoretic mobility (EM) measurements. Sexual transmission events provide a more acidic environment for HIV-1 compared to dissemination and spread of infection occurring in blood or lymph node. We hypothesize functional, trimeric Env behaves differently than monomeric forms.ResultsThe dynamic electrophoretic fingerprint of trimeric gp140 revealed a change in EM from strongly negative to strongly positive as pH increased from that of the lower female genital tract (pHx) to that of the blood (pHy). Similar findings were observed using a trimeric influenza Haemagglutinin (HA) glycoprotein, indicating that this may be a general attribute of trimeric viral envelope glycoproteins. These findings were supported by computationally modeling the surface charge of various gp120 and HA crystal structures. To identify the behavior of the infectious agent and its target cells, EM measurements were made on purified whole HIV-1 virions and primary T-lymphocytes. Viral particles had a largely negative surface charge, and lacked the regions of positivity near neutral pH that were observed with trimeric Env. T cells changed their surface chemistry as a function of activation state, becoming more negative over a wider range of pH after activation. Soluble recombinant CD4 (sCD4) was found to be positively charged under a wide range of conditions. Binding studies between sCD4 and gp140 show that the affinity of CD4-gp140 interactions depends on pH.ConclusionsTaken together, these findings allow a more complete model of the electrochemical forces involved in HIV-1 Env functionality. These results indicate that the influence of the localized environment on the interactions of HIV with target cells are more pronounced than previously appreciated. There is differential chemistry of trimeric, but not monomeric, Env under conditions which mimic the mucosa compared to those found systemically. This should be taken into consideration during design of immunogens which targets virus at mucosal portals of entry.

Analyzing dynamical simulations of intrinsically disordered proteins using spectral clustering

Continuing improvements in algorithms and computer speeds promise that an increasing number of biomolecular phenomena can be simulated by molecular dynamics to produce accurate ldquotrajectoriesrdquo of their molecular motions on the nanosecond to microsecond time scale. An important target for such simulations will be non-equilibrium biochemical processes, such as protein folding, but existing tools for analyzing molecular dynamics trajectories are not well suited to non-equilibrium processes and progress will require improvements in tools for classifying the range and types of dynamics exhibited by these systems. An extreme example of a non-equilibrium biochemical process is the function of ldquointrinsically disorderedrdquo proteins - proteins that function without ever folding into a unique structure. In this paper, we demonstrate the use of spectral clustering methods to analyze the data produced from simulations of several forms from one class of intrinsically disordered proteins, the phenylalanine-glycine nucleoporins (FG-Nups). We explain why such methods are well-suited for the data produced by our simulations and show that clustering methods provide a direct, quantitative measure of how effectively single simulations independently sample regions of structural phase space. Moreover, our clustering results show distinct dynamical behavior in different forms of the FG-Nups, which may provide insights into their biological function.

Switch loop flexibility affects substrate transport of the AcrB efflux pump

The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.

Emergency Automated Response System (EARS)

This paper presents a comparison of two versions of an Emergency Automated Response System (EARS), a fully manual version and a partially automated version. User evaluations involving both versions of the system were conducted using a low workload task and a high workload task. The results indicate that the automation employed by the partially automated system decreased overall response time and perceived workload for both tasks, but accuracy decreased and response times increased from low workload to high workload with both versions.

Computational Modeling of pH-dependent gp120-CD4 Interactions in Founder and Chronic HIV Strains

Human Immunodeficiency Virus has been studied for several decades, but a consistently effective vaccine has not yet been produced. While most experimental and computational work in this area has been performed under slightly basic conditions (eg. blood/plasma), viral transmission events often occur under highly acidic conditions (eg. vaginal mucosa). Environmental pH can greatly affect protein structure and epitope exposure to either inhibit or facilitate transmission. We develop a computational pipeline for analyzing the pH sensitivity of protein-protein interactions, and use the pipeline to analyze the transmission-critical interaction between the HIV gp120 and host CD4 proteins. The interaction between gp120 and CD4 is found to be stronger at low pH for all strains tested, which is consistent with previous work and supports the accuracy of the introduced pipeline. Also, early transmitted founder (TF) strains are found to generally bind CD4 better at low pH and are more pH sensitive than systemically circulating chronic control (CC) strains.

Molecular dynamics simulations of highly charged green fluorescent proteins

A recent experimental study showed that green fluorescent proteins (GFPs) that have been mutated to have ultra-high positive or negative net charges, retain their native structure and fluorescent properties while gaining resistance to aggregation under denaturing conditions. These proteins also provide an ideal test case for studying the effects of surface charge on protein structure and dynamics. Classical molecular dynamics (MD) simulations have been performed on the near-neutral wildtype GFP and mutants with net charges of −29 and +35. The resulting trajectories were analysed to quantify differences in structure and dynamics between the three GFPs. This analysis shows that all three proteins are stable over the MD trajectory, with the near-neutral wild type GFP exhibiting somewhat more flexibility than the positive or negative GFP mutants, as measured by the order parameter and changes in ϕ–ψ angles. There are more dramatic differences in the properties of the water and counter ions surrounding the proteins. The water diffusion constant near the protein surface is closer to the value for bulk water in the positively charged GFP than in the other two proteins. Additionally, the positively charged GFP shows a much greater clustering of the counter ions (Cl−) near its surface than corresponding counter ions (Na+) near the negatively charged mutant.