Determinants Of Substrate Specificity Research Articles

Molecular dynamics simulations shed light into the donor substrate specificity of vertebrate poly-alpha-2,8-sialyltransferases ST8Sia IV

BackgroundSialic acids are essential monosaccharides influencing several biological processes and disease states. The sialyltransferases catalyze the transfer of Sia residues to glycoconjugates playing critical roles in cellular recognition and signaling. Despite their importance, the molecular mechanisms underlying their substrate specificity, especially between different organisms, remain poorly understood. Recently, the human ST8Sia IV, a key enzyme in the synthesis of polysialic acids, was found to accept only CMP-Neu5Ac as a sugar-donor, whereas the whitefish Coregonus maraena enzyme showed a wider donor substrate specificity, accepting CMP-Neu5Ac, CMP-Neu5Gc, and CMP-Kdn. However, what causes these differences in donor substrate specificity is unknown. MethodsComputational approaches were used to investigate the structural and biochemical determinants of the donor substrate specificity in ST8Sia IV. Accurate structural models of the human and fish ST8Sia IV catalytic domains and their complexes with three sialic acid donors (CMP-Neu5Ac, CMP-Neu5Gc, and CMP-Kdn) were generated. Subsequently, molecular dynamics simulations were conducted to analyze the stability and interactions within these complexes and identify differences in complex stability and substrate binding sites between the two ST8Sia IV. ResultsOur MD simulations revealed that the human enzyme effectively stabilizes CMP-Neu5Ac, whereas CMP-Neu5Gc and CMP-Kdn are unstable and explore different conformations. In contrast, the fish ST8Sia IV stabilizes all three donor substrates. Based on these data, we identified the key interacting residues for the different Sias parts of the substrate donors. General significanceThis work advances our knowledge of the enzymatic mechanisms governing sialic acid transfer, shedding light on the evolutionary adaptations of sialyltransferases.

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4–3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

Structural and functional validation of a highly specific Smurf2 inhibitor.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.

Using explainable machine learning to uncover the kinase-substrate interaction landscape.

Phosphorylation, a post-translational modification regulated by protein kinase enzymes, plays an essential role in almost all cellular processes. Understanding how each of the nearly 500 human protein kinases selectively phosphorylates their substrates is a foundational challenge in bioinformatics and cell signaling. Although deep learning models have been a popular means to predict kinase-substrate relationships, existing models often lack interpretability and are trained on datasets skewed toward a subset of well-studied kinases. Here we leverage recent peptide library datasets generated to determine substrate specificity profiles of 300 serine/threonine kinases to develop an explainable Transformer model for kinase-peptide interaction prediction. The model, trained solely on primary sequences, achieved state-of-the-art performance. Its unique multitask learning paradigm built within the model enables predictions on virtually any kinase-peptide pair, including predictions on 139 kinases not used in peptide library screens. Furthermore, we employed explainable machine learning methods to elucidate the model's inner workings. Through analysis of learned embeddings at different training stages, we demonstrate that the model employs a unique strategy of substrate prediction considering both substrate motif patterns and kinase evolutionary features. SHapley Additive exPlanation (SHAP) analysis reveals key specificity determining residues in the peptide sequence. Finally, we provide a web interface for predicting kinase-substrate associations for user-defined sequences and a resource for visualizing the learned kinase-substrate associations. All code and data are available at https://github.com/esbgkannan/Phosformer-ST. Web server is available at https://phosformer.netlify.app.

Characterization of PglJ, a Glycosyltransferase in the Campylobacter concisus N-Linked Protein Glycosylation Pathway that Expands Glycan Diversity.

The Campylobacter genus of Gram-negative bacteria is characterized by the expression of N-linked protein glycosylation (pgl) pathways. As Campylobacter concisus is an emerging human pathogen, a better understanding of the variation of the biosynthetic pathways across the genus is necessary to identify the relationships between protein glycosylation and disease. The pgl pathways of C. concisus strains have been reported to diverge from other Campylobacter in steps after the biosynthesis of N-acetylgalactosamine-α1,3-N,N'-diacetylbacillosamine-α-1-diphosphate undecaprenyl (GalNAc-diNAcBac-PP-Und), which is catalyzed by PglC and PglA, a phosphoglycosyltransferase (PGT) and a glycosyltransferase (GT), respectively. Here we characterize the PglJ GTs from two strains of C. concisus. Chemical synthesis was employed to access the stereochemically defined glycan donor substrates, uridine diphosphate N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA) and uridine diphosphate N-acetyl-d-glucosaminuronic acid (UDP-GlcNAcA), to allow biochemical investigation of PglJ. Evidence for the PglJ substrate specificity structural determinants for the C6″ carboxylate-containing sugar was obtained through variant-based biochemical assays. Additionally, characterization of a UDP-sugar dehydrogenase encoded in the pgl operon, which is similar to the Pseudomonas aeruginosa WbpO responsible for the oxidization of a UDP-HexNAc to UDP-HexNAcA, supports the availability of a UDP-HexNAcA substrate for a GT that incorporates the modified sugar and provides evidence for the presence of a HexNAcA in the N-linked glycan. Utilizing sequence similarity network (SSN) analysis, we identified conserved sequence motifs among PglJ glycosyltransferases, shedding light on substrate preferences and offering predictive insights into enzyme functions across the Campylobacter genus. These studies now allow detailed characterization of the later steps in the pgl pathway in C. concisus strains and provide insights into enzyme substrate specificity determinants for glycan assembly enzymes.

Elucidation of the mescaline biosynthetic pathway in peyote (Lophophora williamsii).

Peyote (Lophophora williamsii) is an entheogenic and medicinal cactus native to the Chihuahuan desert. The psychoactive and hallucinogenic properties of peyote are principally attributed to the phenethylamine derivative mescaline. Despite the isolation of mescaline from peyote over 120 years ago, the biosynthetic pathway in the plant has remained undiscovered. Here, we use a transcriptomics and homology-guided gene discovery strategy to elucidate a near-complete biosynthetic pathway from l-tyrosine to mescaline. We identified a cytochrome P450 that catalyzes the 3-hydroxylation of l-tyrosine to l-DOPA, a tyrosine/DOPA decarboxylase yielding dopamine, and four substrate-specific and regiospecific substituted phenethylamine O-methyltransferases. Biochemical assays with recombinant enzymes or functional analyses performed by feeding putative precursors to engineered yeast (Saccharomyces cerevisiae) strains expressing candidate peyote biosynthetic genes were used to determine substrate specificity, which served as the basis for pathway elucidation. Additionally, an N-methyltransferase displaying broad substrate specificity and leading to the production of N-methylated phenethylamine derivatives was identified, which could also function as an early step in the biosynthesis of tetrahydroisoquinoline alkaloids in peyote.

Rational engineering of an elevator-type metal transporter ZIP8 reveals a conditional selectivity filter critically involved in determining substrate specificity

Engineering of transporters to alter substrate specificity as desired holds great potential for applications, including metabolic engineering. However, the lack of knowledge on molecular mechanisms of substrate specificity hinders designing effective strategies for transporter engineering. Here, we applied an integrated approach to rationally alter the substrate preference of ZIP8, a Zrt-/Irt-like protein (ZIP) metal transporter with multiple natural substrates, and uncovered the determinants of substrate specificity. By systematically replacing the differentially conserved residues with the counterparts in the zinc transporter ZIP4, we created a zinc-preferring quadruple variant (Q180H/E343H/C310A/N357H), which exhibited largely reduced transport activities towards Cd2+, Fe2+, and Mn2+ whereas increased activity toward Zn2+. Combined mutagenesis, modeling, covariance analysis, and computational studies revealed a conditional selectivity filter which functions only when the transporter adopts the outward-facing conformation. The demonstrated approach for transporter engineering and the gained knowledge about substrate specificity will facilitate engineering and mechanistic studies of other transporters.

Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing.

The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.

Identification of amino acid substitutions that toggle substrate selectivity of the yeast arsenite transporter Acr3

The Acr3 protein family plays a crucial role in metalloid detoxification and includes members from bacteria to higher plants. Most of the Acr3 transporters studied so far are specific for arsenite, whereas Acr3 from budding yeast also shows some capacity to transport antimonite. However, the molecular basis of Acr3 substrate specificity remains poorly understood. By analyzing randomly generated and rationally designed yeast Acr3 variants, critical residues determining substrate specificity were identified for the first time. Replacement of Val173 with Ala abolished antimonite transport without affecting arsenite extrusion. In contrast, substitution of Glu353 with Asp resulted in a loss of arsenite transport activity and a concomitant increase in antimonite translocation capacity. Importantly, Val173 is located close to the hypothetical substrate binding site, whereas Glu353 has been proposed to participate in substrate binding. Identification of key residues conferring substrate selectivity provides a valuable starting point for further studies of the Acr3 family and may have implications for the development of biotechnological applications in metalloid remediation. Moreover, our data contribute to understanding why members of the Acr3 family evolved as arsenite-specific transporters in an environment of ubiquitously present arsenic and trace amounts of antimony.

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9.

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.

Lysine Deacetylase Substrate Selectivity: Distinct Interaction Surfaces Drive Positive and Negative Selection for Residues Following Acetyllysine.

Lysine acetylation is a post-translational modification that is reversed by lysine deacetylases (KDACs). The goal of this work was to identify determinants of substrate specificity for KDACs, focusing on short-range interactions occurring with residues immediately following the acetyllysine. Using a fluorescence-based in vitro assay, we determined the activity for each enzyme with a limited panel of derivative substrate peptides, revealing a distinct reactivity profile for each enzyme. We mapped the interaction surface for KDAC6, KDAC8, and KDAC1 with the +1 and +2 substrate residues (with respect to acetyllysine) based on enzyme-substrate interaction pairs observed in molecular dynamics simulations. Characteristic residues in each KDAC interact preferentially with particular substrate residues and correlate with either enhanced or inhibited activity. Although nonpolar aromatic residues generally enhanced activity with all KDACs, the manner in which each enzyme interacted with these residues is distinct. Furthermore, each KDAC has distinctive interactions that correlate with lower activity, primarily ionic in nature. KDAC8 exhibited the most diverse and widest range of effects, while KDAC6 was sensitive only to the +1 position and KDAC1 selectivity was primarily driven by negative selection. The substrate preferences were validated for KDAC6 and KDAC8 using a set of peptides derived from known acetylated proteins. Overall, we determined how KDAC6, KDAC8, and KDAC1 achieve substrate specificity with residues following the acetyllysine. These new insights into KDAC specificity will be critical for identifying novel substrates of particular KDACs, designing KDAC-specific inhibitors, and demonstrate a general framework for understanding substrate specificity for other enzyme classes.

Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.

Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry.

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free protein synthesis as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry (BLI) analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

Methotrexate recognition by the human reduced folate carrier SLC19A1.

The human Reduced Folate Carrier (hRFC) is essential for folate uptake into mammalian cells. As a critical substrate in nucleotide biosynthesis and amino acid metabolism, folate is required for cell division. Consequently, cancerous cells rely heavily upon folate for their growth, so specific targeting by anti-folate pharmaceuticals is highly sought after. One such anti-folate chemotherapeutic is methotrexate (MTX), which requires hRFC for uptake into the cell and variants of which can lead to drug resistance and folate deficiency. Unravelling the transport mechanism of hRFC, as well as the basis for its interaction with folates and anti-folate drugs has important implications for chemotherapeutic development and personalized medicine. In our companion poster [“Cryo-EM structure determination of the human reduced folate carrier SLC19A1 in complex with methotrexate”] we detail the biochemical and structural techniques required for elucidating the structure of MTX-bound hRFC (65 kDa). Here we present transport assay data and MD simulations that build upon the structure to identify the features of hRFC that make it capable of transporting a wide range of anions, while also probing the molecular determinants of substrate specificity. This multifaceted study provides not only insight into the transport mechanism of hRFC but also a basis for rational chemotherapeutic drug design.

Multiple phosphorylation of the Cdc48/p97 cofactor protein Shp1/p47 occurs upon cell stress in budding yeast.

The homohexameric p97 complex, composed of Cdc48 subunits in yeast, is a crucial component of protein quality control pathways including ER-associated degradation. The complex acts to segregate protein complexes in an ATP-dependent manner, requiring the engagement of cofactor proteins that determine substrate specificity. The function of different Cdc48 cofactors and how they are regulated remains relatively poorly understood. In this study, we assess the phosphorylation of Cdc48 adaptor proteins, revealing a unique and distinctive phosphorylation pattern of Shp1/p47 that changed in response to TORC1 inhibition. Site-directed mutagenesis confirmed that this pattern corresponded to phosphorylation at residues S108 and S315 of Shp1, with the double-phosphorylated form becoming predominant upon TORC1 inhibition, ER-stress, and oxidative stress. Finally, we assessed candidate kinases and phosphatases responsible for Shp1 phosphorylation and identified two regulators. We found that cells lacking the kinase Mpk1/Slt2 show reduced Shp1 phosphorylation, whereas impaired PP1 phosphatase catalytic subunit (Glc7) activity resulted in increased Shp1 phosphorylation. Overall, these findings identify a phosphoregulation of Shp1 at multiple sites by Mpk1 kinase and PP1 phosphatase upon various stresses.

Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase

CTX-M β-lactamases are prevalent antibiotic resistance enzymes and are notable for their ability to rapidly hydrolyze the extended-spectrum cephalosporin, cefotaxime. We hypothesized that the active site sequence requirements of CTX-M-mediated hydrolysis differ between classes of β-lactam antibiotics. Accordingly, we use codon randomization, antibiotic selection, and deep sequencing to determine the CTX-M active-site residues required for hydrolysis of cefotaxime and the penicillin, ampicillin. The study reveals positions required for hydrolysis of all β-lactams, as well as residues controlling substrate specificity. Further, CTX-M enzymes poorly hydrolyze the extended-spectrum cephalosporin, ceftazidime. We further show that the sequence requirements for ceftazidime hydrolysis follow those of cefotaxime, with the exception that key active-site omega loop residues are not required, and may be detrimental, for ceftazidime hydrolysis. These results provide insights into cephalosporin hydrolysis and demonstrate that changes to the active-site omega loop are likely required for the evolution of CTX-M-mediated ceftazidime resistance.

Interaction of a Homologous Series of Amphiphiles with P-glycoprotein in a Membrane Environment—Contributions of Polar and Non-Polar Interactions

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [125I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P-gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P-gp's substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P-gp's binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.