Lenacapavir (LEN) is a long-acting, highly potent HIV-1 capsid (CA) inhibitor. The evolution of viral variants under the genetic pressure of LEN identified Q67H, N74D, and Q67H/N74D CA substitutions ...as the main resistance associated mutations (RAMs). Here, we determined high-resolution structures of CA hexamers containing these RAMs in the absence and presence of LEN. Our findings reveal that the Q67H change induces a conformational switch, which adversely affects the inhibitor binding. In the unliganded protein, the His67 side chain adopts the closed conformation by projecting into the inhibitor binding pocket and thereby creating steric hindrance with respect to LEN. Upon the inhibitor binding, the His67 side chain repositions to the open conformation that closely resembles the Gln67 side chain in the WT protein. We propose that the switch from the closed conformation to the open conformation, which is needed to accommodate LEN, accounts for the reduced inhibitor potency with respect to the Q67H CA variant. The N74D CA change results in the loss of a direct hydrogen bond and in induced electrostatic repulsions between CA and LEN. The double Q67H/N74D substitutions exhibited cumulative effects of respective single amino acid changes. An examination of LEN binding kinetics to CA hexamers revealed that Q67H and N74D CA changes adversely influenced the inhibitor binding affinity (
) by primarily affecting the dissociation rate constant (
). We used these structural and mechanistic findings to rationally modify LEN. The resulting analog exhibited increased potency against the Q67H/N74D viral variant. Thus, our studies provide a means for the development of second-generation inhibitors with enhanced barriers to resistance.
LEN is an investigational long-acting agent for future HIV-1 treatment regimens. While ongoing clinical trials have highlighted a largely beneficial profile of LEN for the treatment of HIV-1 infected people with limited therapy options, one notable shortcoming is a relatively low barrier of viral resistance to the inhibitor. Cell culture-based viral breakthrough assays identified N74D, Q67H, and N74D/Q67H capsid changes as the main resistance associated mutations (RAMs). N74D and Q67H capsid substitutions have also emerged in clinical trials in some patients who received subcutaneous LEN. Understanding the structural basis behind viral resistance to LEN is expected to aid in the rational development of improved inhibitors with enhanced barriers to resistance. Here, we report high resolution structures of the main drug resistant capsid variants, which provide mechanistic insight into the viral resistance to LEN. We used these findings to develop an improved inhibitor, which exhibited enhanced activity against the viral Q67H/N74D capsid phenotype compared with that of parental LEN.
Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short phenylalanine-glycine (FG) containing peptides with isolated capsid hexamers have ...been characterized, how these cellular factors functionally engage with biologically relevant mature HIV-1 capsid lattices is unknown. Here we show that prion-like low complexity regions (LCRs) enable avid CPSF6, NUP153 and SEC24C binding to capsid lattices. Structural studies revealed that multivalent CPSF6 assembly is mediated by LCR-LCR interactions, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. In infected cells, avid CPSF6 LCR-mediated binding to HIV-1 cores is essential for functional virus-host interactions. The investigational drug lenacapavir accesses unoccupied hydrophobic pockets in the complex to potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results establish previously undescribed mechanisms of virus-host interactions and antiviral action.
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal ...structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target.
Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our results explain the mechanism of drug action and will inform the development of this promising class of small molecules for future use in antiretroviral regimens.
Bacterial esterases are highly versatile enzymes, currently widely used in detergents, biosurfactants, bioemulsifiers and as biocatalysts in paper and food industries. Present work describes ...heterologous expression, purification, and biophysical and biochemical characterization of a halotolerant esterase from
Bacillus licheniformis
(
Bl
EstA).
Bl
EstA preferentially cleaves
pNP
-octanoate and both activity and stability of the enzyme increased in the presence of 2 M NaCl, and also with several organic solvents (ethanol, methanol and DMSO). Furthermore,
Bl
EstA has considerable emulsifying properties, particularly with olive oil as substrate. Our studies also show that the enzyme is monomeric in solution and its small-angle X-ray scattering low-resolution molecular envelope fits well its high-resolution homology model.
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•PxXyn11B is the first crystal structure of a GH11 from Paenibacillus.•X-ray structure of PxXyn11B reveals its molecular architecture and a second binding site (SBS).•MD simulations ...shed light on structural determinants of uncommon PxXyn11B cooperative behavior.•Our results reveal molecular mechanism of SBS communication with the active site in GH11 scaffold settings.
Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-β-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.
•T. composti GH10 xylanase is thermostable, halotolerant and ethanol-resistant.•X-ray structure of TcXyn10A reveals molecular details of xylan recognition.•TcXyn10A improves efficiency of pretreated ...lignocellulosic biomass hydrolysis.•The enzyme liberates xylooligosaccharides from xylan with elevated yields.
Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.
Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-β-mannosidases. These enzymes are involved in mannan degradation, and have a number of ...biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a − 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.
The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a ...diverse set of enzymes including endo‐β‐1,4‐xylanases, β‐xylosidases, acetylxylan esterases, α‐l‐arabinofuranosidases, and α‐glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo‐β‐1,4‐ and β‐1,3‐xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose‐bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.
Structural and biochemical studies were performed to characterize an unusual enzyme from GH11 family which was identified in a compost‐derived consortium. The enzyme has a xylobiohydrolase activity, quite uncommon for members of GH11 family. Structural studies revealed that a lack of ‐3 subsite in the active site of the enzyme restrained a range of its catalytic products, leading to a release of only xylobiose (X2) during xylan hydrolysis.
HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), ...first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.
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A crescente preocupação com o meio-ambiente requer alternativas para o consumo de combustíveis fosseis. O biodiesel, por exemplo, foi desenvolvido como alternativa ao óleo diesel mineral. Sua produção é bastante grande e tende a crescer ainda mais. Entretanto, em sua produção, o biodiesel produz glicerol, que necessita de purificação, em um processo bastante caro. Uma alternativa seria a utilização do glicerol bruto para a produção de outros compostos de maior valor, através de fermentação. Nesse caminho, uma das alternativas seria a produção de 1,3-propanodiol (1,3-PDO), um álcool de alto valor industrial, com diversos usos nas diversas áreas industriais, em especial na produção de um polímero similar ao PET, o PTT. A produção de 1,3-PDO a partir de glicerol é conhecida em alguns micro-organismos, como a Klebsiella pneumoniae, por exemplo. O presente trabalho teve por objetivo construir a via de produção de 1,3-PDO, presente em K. pneumoniae, em Escherichia coli geneticamente modificada, para a produção de 1,3-PDO através de um micro-organismo bastante utilizado na industria, utilizando a engenharia metabólica
The growing concern with the environment requires the use of alternatives to fossil fuels. Biodiesel, for example, was developed as an alternative to mineral diesel. Its production is quite large and likely to grow even more. However, in its production, biodiesel produces glycerol, which needs purification, in a quite expensive process. An alternative would be the use of raw glycerol to produce other compounds of higher value by fermentation. In this way, an alternative would be the production of 1,3-propanediol (1,3-PDO), an alcohol of high industrial value with many uses in several industrial fields, particularly in the production of a polymer similar to PET, the PTT. The production of 1,3-PDO from glycerol is known in some micro-organisms such as Klebsiella pneumoniae, for example. The present study aimed to build the pathway of the production of 1,3-PDO, present in K. pneumoniae, in Escherichia coli genetically modified, for the production of 1,3-PDO by a micro-organism widely used in industry, using metabolic engineering
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