Binding and Movement of Individual Cel7A Cellobiohydrolases on Crystalline Cellulose Surfaces Revealed by Single-molecule Fluorescence Imaging

The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate.     

Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1

Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendall's tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donor's antibodies, and five of these were located within an 18-residue amphipathic helix (alpha2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire alpha2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in alpha2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the alpha2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the alpha2 helix for efficient viral replication and immune avoidance.     

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. © 2014 Wiley Periodicals, Inc.     

The B cell response is redundant and highly focused on V1V2 during early subtype C infection in a Zambian seroconverter

High-titer autologous neutralizing antibody responses have been demonstrated during early subtype C human immunodeficiency virus type 1 (HIV-1) infection. However, characterization of this response against autologous virus at the monoclonal antibody (MAb) level has only recently begun to be elucidated. Here we describe five monoclonal antibodies derived from a subtype C-infected seroconverter and their neutralizing activities against pseudoviruses that carry envelope glycoproteins from 48 days (0 month), 2 months, and 8 months after the estimated time of infection. Sequence analysis indicated that the MAbs arose from three distinct B cell clones, and their pattern of neutralization compared to that in patient plasma suggested that they circulated between 2 and 8 months after infection. Neutralization by MAbs representative of each B cell clone was mapped to two residues: position 134 in V1 and position 189 in V2. Mutational analysis revealed cooperative effects between glycans and residues at these two positions, arguing that they contribute to a single epitope. Analysis of the cognate gp120 sequence through homology modeling places this potential epitope near the interface between the V1 and V2 loops. Additionally, the escape mutation R189S in V2, which conferred resistance against all three MAbs, had no detrimental effect on virus replication in vitro. Taken together, our data demonstrate that independent B cells repeatedly targeted a single structure in V1V2 during early infection. Despite this assault, a single amino acid change was sufficient to confer complete escape with minimal impact on replication fitness.     

Quantifying intramolecular binding in multivalent interactions: a structure-based synergistic study on Grb2-Sos1 complex

Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment.     

Assisted peptide folding by surface pattern recognition

Natively disordered proteins belong to a unique class of biomolecules whose function is related to their flexibility and their ability to adopt desired conformations upon binding to substrates. In some cases these proteins can bind multiple partners, which can lead to distinct structures and promiscuity in functions. In other words, the capacity to recognize molecular patterns on the substrate is often essential for the folding and function of intrinsically disordered proteins. Biomolecular pattern recognition is extremely relevant both in vivo (e.g., for oligomerization, immune response, induced folding, substrate binding, and molecular switches) and in vitro (e.g., for biosensing, catalysis, chromatography, and implantation). Here, we use a minimalist computational model system to investigate how polar/nonpolar patterns on a surface can induce the folding of an otherwise unstructured peptide. We show that a model peptide that exists in the bulk as a molten globular state consisting of many interconverting structures can fold into either a helix-coil-helix or an extended helix structure in the presence of a complementary designed patterned surface at low hydrophobicity (3.7%) or a uniform surface at high hydrophobicity (50%). However, we find that a carefully chosen surface pattern can bind to and catalyze the folding of a natively unfolded protein much more readily or effectively than a surface with a noncomplementary or uniform distribution of hydrophobic residues.     

Temperature Dependence of Water Interactions with the Amide Carbonyls of α-Helices

Hydration is a key determinant of the folding, dynamics, and function of proteins. In this study, temperature-dependent Fourier transform infrared (FTIR) spectroscopy combined with singular value decomposition (SVD) and global fitting were used to investigate both the interaction of water with α-helical proteins and the cooperative thermal unfolding of these proteins. This methodology has been applied to an isolated α-helix (Fs peptide) and to globular α-helical proteins including the helical subdomain and full-length villin headpiece (HP36 and HP67). The results suggest a unique IR signature for the interaction of water with the helical amide carbonyl groups of the peptide backbone. The IR spectra indicate a weakening of the net hydrogen bond strength of water to the backbone carbonyls with increasing temperature. This weakening of the backbone solvation occurs as a discrete transition near the maximum of the temperature-dependent hydrophobic effect, not a continuous change with increasing temperature. Possible molecular origins of this effect are discussed with respect to previous molecular dynamics simulations of the temperature-dependent solvation of the helix backbone.     

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.     

A signature in HIV-1 envelope leader peptide associated with transition from acute to chronic infection impacts envelope processing and infectivity

Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.