An official website of the United States government
The highly conserved protease enzyme from SARS-CoV-2 (MPro) is crucial for viral replication and is an attractive target for the design of novel inhibitory compounds. MPro is known to be conformationally flexible and has been stabilized in an extended conformation in a complex with a novel nanobody (NB2B4), which inhibits the dimerization of the enzyme via binding to an allosteric site. However, the energetic contributions of the nanobody residues stabilizing the MPro/nanobody interface remain unresolved. We probed these residues using all-atom MD simulations in combination with alchemical free energy calculations by studying the physical residue–residue interactions and discovered the role of hydrophobic and electrostatic interactions in stabilizing the complex. Specifically, we found via mutational analysis that three interfacial nanobody residues (Y59, R106, and L109) contributed significantly, two residues (L107 and P110) contributed moderately, and two residues (H112 and T113) contributed minimally to the overall binding affinity of the nanobody. We also discovered that the nanobody affinity could be enhanced via a charge-reversal mutation (D62R) that alters the local interfacial electrostatic environment of this residue in the complex. These findings are potentially useful in designing novel synthetic nanobodies as allosteric inhibitors of MPro.
more »
« less
Hydrophobic Residues Promote Interfacial Activation of Candida rugosa Lipase: A Study of Rotational Dynamics
Microbial lipases constitute a class of biocatalysts with the ability to cleave ester linkages of long-chain triglycerides. This property makes them particularly attractive for industrial applications ranging from food processing to pharmaceutical preparation. Among such enzymes, Candida rugosa lipase (CRL) is one of the most frequently used in biotransformation. A notable feature of CRL, among many lipases, is its propensity for interfacial activation: these enzymes exhibit elevated catalytic rates when acting at the interface between aqueous and hydrophobic phases. Notably, this phenomenon can be attributed to the presence of a mobile lid domain, which in its closed state occludes the enzyme active site. To advance our understanding of interfacial activation, we explore the dynamics of CRL rotation at the octane–water interface in this work. To do so, we employ molecular dynamics and umbrella sampling to evaluate the free energy of rotation of the enzyme at the interface. We identify a global minimum in the rotational landscape that coincides with lid opening at the interface. Additionally, we investigate the role of surface residues outside the lid domain as they serve to instigate rotation of the lid toward the aqueous phase. In doing so, we identify a patch of leucine residues which when mutated to glycine impose a barrier to rotation that maintains the enzyme in the inactive (closed lid) state on the order of 1 μs. Importantly, this study presents a novel quantification of the rotational free energy corresponding to CRL lid opening at the octane–water interface. The accompanying mutagenesis study likewise clarifies the role of hydrophobic surface residues in the transition. As such, this work provides valuable insight into the phenomenon of interfacial activation that might open up new avenues for manipulating the microenvironment of industrially relevant lipases, affording enhanced control over the enzyme state.
more »
« less
- PAR ID:
- 10537169
- Publisher / Repository:
- ACS Journals
- Date Published:
- Journal Name:
- Langmuir
- ISSN:
- 0743-7463
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an important role in both cellular physiology (endosome acidification) and diseases associated with tissue acidosis (acid-induced cell death). Despite the available structural information, how proton binding in the extracellular domain (ECD) leads to PAC channel opening remains largely unknown. Here, through comprehensive mutagenesis and electrophysiological studies, we identified several critical titratable residues, including two histidine residues (H130 and H131) and an aspartic acid residue (D269) at the distal end of the ECD, together with the previously characterized H98 at the transmembrane domain–ECD interface, as potential pH sensors for human PAC. Mutations of these residues resulted in significant changes in pH sensitivity. Some combined mutants also exhibited large basal PAC channel activities at neutral pH. By combining molecular dynamics simulations with structural and functional analysis, we further found that the β12 strand at the intersubunit interface and the associated “joint region” connecting the upper and lower ECDs allosterically regulate the proton-dependent PAC activation. Our studies suggest a distinct pH-sensing and gating mechanism of this new family of ion channels sensitive to acidic environment.more » « less
-
null (Ed.)Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.more » « less
-
Abstract Charging of interfaces between water and hydrophobic media is a mysterious feature whose nature and origin have been under debate. Here, we investigate the fundamentals of the interfacial behaviors of water by employing opto-thermophoretic tweezers to study temperature-gradient-induced perturbation of dipole arrangement at water/oil interfaces. With surfactant-free perfluoropentane-in-water emulsions as a model interface, additional polar organic solvents are introduced to systematically modify the structural aspects of the interface. Through our experimental measurements on the thermophoretic behaviors of oil droplets under a light-generated temperature gradient, in combination with theoretical analysis, we propose that water molecules and mobile negative charges are present at the water/oil interfaces with specific dipole arrangement to hydrate oil droplets, and that this arrangement is highly susceptible to the thermal perturbation due to the mobility of the negative charges. These findings suggest a potential of opto-thermophoresis in probing aqueous interfaces and could enrich understanding of the interfacial behaviors of water.more » « less
-
The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon–carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology, and the structure and mechanism of CylK are unknown. Here, we report X-ray crystal structures of CylK, revealing a distinctive fusion of a Ca 2+ -binding domain and a β-propeller fold. We use a mutagenic screening approach to locate CylK’s active site at its domain interface, identifying two residues, Arg105 and Tyr473, that are required for catalysis. Anomalous diffraction datasets collected with bound bromide ions, a product analog, suggest that these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicate Asp440 in activating the nucleophilic aromatic ring. Bioinformatic analysis of CylK homologs from other cyanobacteria establishes that they conserve these key catalytic amino acids, but they are likely associated with divergent reactivity and altered secondary metabolism. By gaining a molecular understanding of this unusual biosynthetic transformation, this work fills a gap in our understanding of how alkyl halides are activated and used by enzymes as biosynthetic intermediates, informing enzyme engineering, catalyst design, and natural product discovery.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1021/acs.langmuir.4c02174
