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Certain immune-driven mutations in HIV-1, such as those arising in p24 Gag , decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24 Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) ( P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles ( P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24 Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.

The original map of mammalian cytochrome P450 (CYP450) residues involved in substrate recognition was prepared for the CYP2 family by Gotoh in 1992 by manual alignment of mammalian CYP450 residues with substrate recognition site (SRS) residues manually delimited from a bacterial cytochrome P450-substrate complex. Using modern structural bioinformatics tools, we have identified CYP450-ligand interactions in mammalian complexes to create a "X-ray structures" SRS map. In a parallel approach, we have built a "docking" SRS map by successful docking of 868 known substrates of 10 mammalian CYP450 isoforms and analysis of contacts made in docking solutions. We subsequently combined these maps to create a unified description of SRSs. The new map largely agrees with the original map by Gotoh with the six original SRS regions appearing in similar locations along the CYP450 sequence as in Gotoh's map. However, important differences also occur: Two new SRS regions appear before SRS1 and we have assigned them as SRS1'a and SRS1'b; SRS1 is much bigger in our map than in Gotoh's (49 aligned positions versus 28); & SRS2 and SRS3 are co-joined in our map to give a single large SRS region (60 aligned positions) we have designated as SRS(2,3), in contrast to the 9 and 10 aligned positions individually covered by SRS2 and SRS3 respectively in Gotoh's original map. These differences result in the SRS zone covering 33 % of the mammalian CYP450 sequence in our map as opposed to 16 % in Gotoh's map.

The study of the protein–protein interactions (PPIs) of unique ORFs is a strategy for deciphering the biological roles of unique ORFs of interest. For uniform reference, we define unique ORFs as those for which no matching protein is found after PDB-BLAST search with default parameters. The uniqueness of the ORFs generally precludes the straightforward use of structure-based approaches in the design of experiments to explore PPIs. Many open-source bioinformatics tools, from the commonly-used to the relatively esoteric, have been built and validated to perform analyses and/or predictions of sorts on proteins. How can these available tools be combined into a protocol that helps the non-expert bioinformaticist researcher to design experiments to explore the PPIs of their unique ORF? Here we define a pragmatic protocol based on accessibility of software to achieve this and we make it concrete by applying it on two proteins—the ImuB and ImuA’ proteins from Mycobacterium tuberculosis. The protocol is pragmatic in that decisions are made largely based on the availability of easy-to-use freeware. We define the following basic and user-friendly software pathway to build testable PPI hypotheses for a query protein sequence: PSI-PRED → MUSTER → metaPPISP → ASAView and ConSurf. Where possible, other analytical and/or predictive tools may be included. Our protocol combines the software predictions and analyses with general bioinformatics principles to arrive at consensus, prioritised and testable PPI hypotheses.

In this work, a model for the interaction between CYP2B4 and the FMN domain of rat P450-oxidoreductase is built using as template the structure of a bacterial redox complex. Amino acid residues identified in the literature as cytochrome P450 (CYP)-redox partner interfacial residues map to the interface in our model. Our model supports the view that the bacterial template represents a specific electron transfer complex and moreover provides a structural framework for explaining previous experimental data. We have used our model in an exhaustive search for complementary pairs of mammalian CYP and P450-oxidoreductase (POR) charge clusters. We quantitatively show that among the previously defined basic clusters, the 433K-434R cluster is the most dominant (32.3% of interactions) and among the acidic clusters, the 207D-208D-209D cluster is the most dominant (29%). Our analysis also reveals the previously not described basic cluster 343R-345K (16.1% of interactions) and 373K (3.2%) and the acidic clusters 113D-115E-116E (25.8%), 92E-93E (12.9%), 101D (3.2%) and 179E (3.2%). Cluster pairings among the previously defined charge clusters include the pairing of cluster 421K-422R to cluster 207D-208D-209D. Moreover, 433K-434R and 207D-208D-209D, respectively the dominant positively and negatively charged clusters, are uncorrelated. Instead our analysis suggests that the newly identified cluster 113D-115E-116E is the main partner of the 433K-434R cluster while the newly described cluster 343R-345K is correlated to the cluster 207D-208D-209D.

We have used maximum-likelihood models of codon substitution to investigate the role of adaptive evolution in the evolution of cytochrome P450 (CYP) sequences. Evidence for the operation of adaptive evolution in the evolution of rat CYP2C, rabbit CYP2C, rat CYP2D, human CYP3A and rabbit CYP4A was observed. The absence of signal in rat CYP2B, rat CYP3A, human CYP2C and monkey CYP2C suggests that the adaptive evolution did not operate in the evolution of these cytochromes. Our results show identical adaptive evolution patterns for rabbit (lagomorpha) and rat (rodentia) CYP2C. The absence of signal for adaptive evolution in primate CYP2C suggests that the identical rat and rabbit CYP2C patterns arose in the last common ancestor of rodentia and lagomorpha. Furthermore, we have found statistically significant association of sites under adaptive evolution and Gotoh's substrate recognition sites in rat and rabbit CYP2C (5%), human CYP3A and rat CYP2D (10%). From these correlations, the given substrate-dependent nature of differences in CYP substrate-specificity profiles and differences in cytochrome active-site residues, we hypothesize that the most likely role of adaptive evolution in the evolution of cytochrome P450 substrate specificities was to fix mutations that permitted an increased number of binding modes (thereby expanding the substrate repertoire). The pattern of adaptive evolution observed in this work is consistent with results from microsomal studies in which CYP2C isoforms are responsible for most of the metabolism of foreign compounds in rat and rabbit, and CYP3A isoforms play the same role in humans.