Biology subjects

Schiffer, C. A.

Publications and source records attributed to Schiffer, C. A..

5 recordsLinked to original sources

bioRxiv2018

Structural Adaptation of Darunavir Analogs Against Primary Resistance Mutations in HIV-1 Protease

HIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82 and I84 that line the S1/S1 pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogs to primary S1 mutations. The analogs had modifications at the hydrophobic P1 moiety to better occupy the unexploited space in the S1 pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analog with the largest P1 moiety, indicating interdependency between the S1 subsite and the flap region. Collective analysis of protease-inhibitor van der Waals (vdW) interactions in the crystal structures using principle component analysis indicated I84V mutation underlying the largest variation in the vdW contacts. Interestingly, the principle components were able to distinguish inhibitor identity and relative potency solely based on vdW interactions of active site residues in the crystal structures. Our results reveal the interplay between inhibitor P1 moiety and primary S1 mutations, as well as suggesting a novel method for distinguishing the interdependence of resistance through principle component analyses.

molecular biology↗

bioRxiv2018

HIV-1 Protease Uses Bi-Specific S2/S2’ Subsites To Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PR) have a unique specificity that allows cleavage of sites with or without a P1 proline. A P1 proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 iterations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1-P2), with two complementary sets of rules. When P1 is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2 amino acid interacts with a hydrophobic region of the S2 subsite. When P1 is not proline, the orientations of the P2 and P2 side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite while the P2 amino acid usually engages hydrophilic amino acids in the S2 subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2 subsites to accommodate the steric effects imposed by a P1 proline on the orientation of P2 and P2 substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

biochemistry↗

bioRxiv2018

Constrained mutational sampling of amino acids in HIV-1 protease evolution

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution are interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available dataset of 32,163 sequenced isolates from drug-naive individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple base mutations in HIV-1 protease is strongly influenced by mutational sampling.

evolutionary biology↗

bioRxiv2018

Structural analysis of the active site and DNA binding of human cytidine deaminase APOBEC3B

APOBEC3s proteins (A3s), a family of human cytidine deaminases, protect the host cell from endogenous retro-elements and exogenous viral infections by introducing hypermutations. However, the ability to mutate genomic DNA makes A3s a potential cancer source. Of the 7 human A3s, A3B has been implicated as an endogenous cause for multiple cancers. Despite overall similarity, A3s have distinct deamination activity with A3B among the least catalytically active. Over the past few years, several structures of apo as well as DNA-bound A3 proteins have been determined. These structures revealed the molecular determinants of nucleotide specificity and the importance of the loops around the active site in DNA binding. However, for A3B, the structural basis for regulation of deamination activity and the role of active site loops in coordinating DNA had remained unknown. In this study, using a combination of advanced molecular modelling followed by experimental mutational analysis and dynamics simulations, we investigated molecular mechanism of A3B regulating activity and DNA binding. We identified a unique auto-inhibited conformation of A3B that restricts access and binding of DNA to the active site, mainly due to the extra PLV residues in loop 1. We modelled DNA binding to fully native A3B and found that Arg211 in the arginine patch of loop1 is the gatekeeper while Arg212 stabilizes the bound DNA. This model also identified the critical residues for substrate specificity, especially at the -1 position. Our results reveal the structural basis for relatively lower catalytic activity of A3B and provide opportunities for rational design of inhibitors that specifically target A3B to benefit cancer therapeutics.

biochemistry↗

bioRxiv2017

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, substrate secondary structure, and pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), and found that A3A binds RNA in a sequence specific manner. Furthermore, A3A bound tighter to its substrate binding motif when in a loop compared to linear oligonucleotide. Our results suggest that the A3A affinity and preference for substrate is modulated by the structure of DNA, and not just its chemical identity. Analysis of previously published co-crystal structures of A3A bound to ssDNA in light of the above findings directed the proposal of a new model for the molecular mechanism underlying A3A sequence preference. On a broader scale, the results of this work not only provide key insights into the mechanism of A3s beneficial roles in the cell, especially in viral restriction, but also into A3s deleterious activity such as in the development of cancer.

biochemistry↗