Biochemical Journal

Lanthipeptides are one of the largest classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). The coi biosynthetic gene cluster (BGC) from Streptomyces coelicolor A3(2) encodes a canonical class I lanthipeptide dehydratase (CoiB) and cyclase (CoiC), a bifunctional enzyme (CoiSA) with an O-methyltransferase (MT) and glutamyl lyase (GL) domain, and a protein of unknown function (CoiH). The product of the coi BGC was recently shown to impart anti-phage activity, but its structure is still unresolved. Previous work investigated the regioselectivity of the GL domains in CoiB and CoiSA and the stereochemistry of the cyclized precursor peptide, but the function of CoiH was not addressed. In this study, co-expression of the peptide CoiA1 with CoiBCSAH resulted in a +16 Da addition on the cyclized peptide compared to when CoiH was omitted. LC-MS/MS analysis indicated that this modification occurred in the first thioether ring. A combination of site-directed mutagenesis, comparison of linear and cyclized peptide substrates, hydrogen peroxide (H2O2) treatment, and collision-induced dissociation (CID) mass spectrometric analysis suggested that the sulfur atom in the first methyllanthionine was oxidized to a sulfoxide group by CoiH. This hypothesis was confirmed by NMR analysis. CoiH represents a previously uncharacterized oxygenase family catalyzing sulfoxide formation. Structure prediction tools suggest a novel enzyme fold without obvious metal or cofactor binding sites, raising the possibility that CoiH is a cofactor independent oxidation enzyme.

SUMOylation is an essential eukaryotic ubiquitin-like post-translational modification that plays a central role in the regulation of various nuclear processes and stress responses. It canonically occurs at lysine residues within {Psi}KXE consensus motifs that lie in intrinsically disordered regions or loops and interact specifically with the SUMO-conjugating E2 enzyme UBC9. However, many detected SUMOylation sites are found within structured domains, and it remains unclear how these are recognised by UBC9. Here, we investigated the SUMOylation of Lys43 in the BTB domain of human ZBTB38 (ZBTB38BTB), a lysine located within a rigid {beta}-sheet. By combining X-ray crystallography, structural prediction, and in-vitro UBC9 interaction and SUMOylation assays, we show that ZBTB38BTB possesses a dedicated surface that recapitulates the spatial arrangement of residues found in canonical linear consensus motifs. This surface binds UBC9 with mid-micromolar affinity and is predicted to position Lys43 in its active site for efficient SUMOylation. Structural modelling and sequence analyses suggest that this property is shared by BTB domains of five members (10%) of the ZBTB-protein family across vertebrates, revealing a previously unrecognised property of a subset of ZBTBBTB domains. Kinetic analyses reveal that, under the reaction conditions used, the catalytic efficiency of ZBTB38BTB and ZBTB33BTB SUMOylation are closely comparable to that of the C-terminal domain of RANGAP1, the best-characterised and most efficiently SUMOylated substrate known. This defines a new class of inherently efficient, E3 ligase-independent SUMOylation substrates beyond RANGAP1 and suggests that structural pre-organisation of the acceptor lysine and its environment may promote productive UBC9 engagement. Lastly, we demonstrate the presence of higher-molecular-weight, modified forms of ZBTB38 in human cells, consistent with SUMOylation. Together, these results provide a biochemical basis for interpreting existing and designing future studies on the functional impact of ZBTB SUMOylation. More broadly, our findings offer insights into the determinants of efficient SUMOylation, and may facilitate the identification of further inherently efficient targets, and, potentially, the design of SUMOylation modulators.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that initiates ubiquitin-dependent mitophagy and is mutated in early-onset Parkinson's disease. Despite extensive characterization of insect PINK1 orthologues, obtaining soluble and catalytically active recombinant Homo sapiens PINK1 (HsPINK1) has remained challenging, limiting its biochemical and structural investigation. Here, we identify an evolutionary divergence in the mechanisms supporting PINK1 stability and activity. Comparative analysis of 48 metazoan PINK1 orthologues revealed that predicted binding to the mitochondrial import receptor TOM20 is predominantly a vertebrate feature and coincides with divergence of the PINK1 N- and C-terminal extensions (NTE and CTE). Consistent with this distinction, invertebrate PINK1 orthologues were purified as active kinases, whereas vertebrate orthologues lacked detectable ubiquitin kinase activity. NMR spectroscopy demonstrated that humanization of ten residues within the Tribolium castaneum (Tc)PINK1 NTE-CTE interface is sufficient to confer direct binding to human TOM20, while co-expression with TOM20 increased the activity of recombinant HsPINK1. Guided by these evolutionary differences, we engineered a chimeric PINK1 containing a predominantly human kinase domain supported by Tc-derived NTE-CTE elements. The resulting kinase was catalytically active and required compatible interactions between the NTE-CTE and kinase C-lobe for activity. The chimera enabled characterization of PINK1 variants and revealed R152W as a likely pathogenic variant. Together, our findings support a model in which the NTE and CTE act as intramolecular determinants of PINK1 kinase stability and suggest that vertebrate PINK1 has evolved an increased dependence on the mitochondrial import machinery to maintain a functional state.

Phosphatidic acid (PA) phosphatase (PAP) is an enzyme that plays a major role in lipid signaling by controlling the cellular levels of two lipid secondary messengers: its substrate, PA, and its product, diacylglycerol. Two types of mammalian PAPs have been reported to date. Type 1 PAP (PAP1) is an Mg2+-dependent, N-ethylmaleimide (NEM)-sensitive cytosolic enzyme (EC 3.1.3.4), whereas type 2 PAP (PAP2), also known as phospholipid phosphate (PLPP) (EC 3.1.3.113), is an Mg2+-independent, NEM-insensitive transmembrane protein. PAP2 also hydrolyzes other bioactive lipids such as lyso-PA (LPA), sphingosine-1-phosphate (S1P), and ceramide-1-phosphate (C1P). Here, we purified human phosphatase orphan 2 (PHOSPHO2), a putative cytosolic phosphatase containing a haloacid dehalogenase-like domain, and characterized its enzymological properties in vitro. Purified PHOSPHO2 displays Mg2+-dependent, NEM-sensitive phosphatase activities toward PA, LPA, S1P, C1P, and glycerol-3-phosphate (G3P) in vitro. Moreover, PHOSPHO2 showed substrate selectivity for PA molecular species containing shorter saturated fatty acids such as lauric acid and myristic acid, or polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid. The PAP activity of PHOSPHO2, but not its other phosphatase activities, was strongly enhanced in the presence of phosphatidylcholine and phosphatidylethanolamine, major components of the cell membranes. These results indicate that mammalian PHOSPHO2 is a novel cytosolic PLPP that primarily functions as a PAP on cytoplasm-facing membranes.

Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKII, which could be suitable for subsequent biochemical and structural studies.

Calcineurin (Cn) interacts with the Na+/H+-exchanger NHE1 through its calcineurin-binding domain (NHE1-CnBD). How activation of full-length Cn influences this interaction and its functional consequences has remained unclear. Here, we quantified the interaction between full-length human Cn and NHE1-CnBD in distinct activation states using fluorescence anisotropy and quantitative size-exclusion chromatography. Ca2+/calmodulin activation increased Cn affinity for NHE1-CnBD from submicromolar to tens of nanomolar affinity and stabilized the complex. NHE1-CnBD outcompeted the immunosuppressive cyclosporin A/cyclophilin A complex for Cn binding and selectively inhibited dephosphorylation of a phosphopeptide substrate while enhancing hydrolysis of p-nitrophenyl phosphate. These findings identify NHE1 as a high-affinity, substrate-competitive Cn regulator that blocks substrate recognition rather than catalysis, providing a mechanistic framework for reciprocal regulation between Cn and NHE1.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide, yet therapeutic options remain limited. TMPRSS6, a liver serine protease best known for its role in iron homeostasis, has recently emerged as a potential therapeutic target for MASLD. However, the molecular mechanisms linking TMPRSS6 to hepatic lipid metabolism remain incompletely understood. To identify novel TMPRSS6 substrates, we performed extracellular proteomic analyses of TMPRSS6-overexpressing cells. Among the proteins identified, {beta}-klotho (KLB), a co-receptor required for FGF19 and FGF21 signaling, emerged as a compelling candidate substrate. We demonstrate that TMPRSS6 interacts with KLB and promotes its proteolytic shedding in a catalytic activity-dependent manner. Functionally, TMPRSS6 reduced full-length KLB abundance at the cell surface and attenuated FGF19-dependent FGFR4 signaling in a heterologous expression system. Together, these findings identify KLB as a novel functional substrate of TMPRSS6, providing a mechanistic framework through which this protease may influence hepatic lipid metabolism. These results provide a rationale for investigating the regulation of KLB and other candidate substrates by TMPRSS6 in physiological models and further support its evaluation as a therapeutic target for MASLD.

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

Bordetella pertussis, the causative agent of whooping cough, is a reemerging public health threat. While the Tripartite Tricarboxylate Transporter (TTT) system BctCBA was previously implicated solely in citrate uptake, we demonstrate that the solute-binding protein BctC specifically binds citrate chelated with Zn{superscript 2} and Ni{superscript 2}. To elucidate the molecular mechanism of this interaction, we determined the crystal structures of BctC in three states: apo, open, and closed (citrate-zinc-bound), defining the structural determinants for metal-citrate recognition. Comparative analyses suggest that citrate-mediated divalent cation binding is a widespread feature among bacterial TTT homologs. Finally, in silico modeling of the full BctCBA complex predicts an elevator-type transport mechanism. Together, these findings redefine the functional scope of BctCBA, revealing a sophisticated strategy by which B. pertussis exploits organic chelators to acquire essential trace metals during infection.

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron-sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD- and NAD(P)+-interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984_05830, which is annotated as a sporulation protein but possesses all six predicted cofactor-interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin-dependent reductase activity, with a wild-type kcat of 0.007 min-1--a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome-wide screen further revealed a repertoire of ferredoxin-like carriers, suggesting that C. pasteurianum distributes hydrogen-derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor-interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186-199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.

Schistosoma mansoni and its schistosome relatives are parasitic worms that impose a substantial disease burden on human populations and livestock. On the rationale that calcium signalling is a critical process in multicellular organisms, we have examined wildtype and engineered S. mansoni STIM and ORAI-- orthologues of STIM and ORAI known in mammals and other species for their central role in cellular calcium signalling-- by imaging their localization, interactions, and contribution to ion currents and calcium influx in living cells. The ER membrane protein S. mansoni STIM recapitulates the essential functions of mammalian STIM1, namely, calcium-sensing by its ER-luminal domain, targeting to ER-plasma membrane junctions through interactions with the plasma membrane and with plasma membrane S. mansoni ORAI channels, and an ability to gate the S. mansoni ORAI channel. S. mansoni ORAI is a plasma membrane calcium channel that exhibits striking parallels with mammalian ORAI1 in its pore architecture and gating mechanism. The schistosome and human proteins are not completely interchangeable, however, and schistosome-human ORAI chimeras point to a special role of the ORAI N terminus in channel gating. Importantly, we demonstrate pharmacological differences between the schistosome and human channels that may offer an opportunity for selective therapeutic targeting of schistosome STIM-ORAI-dependent calcium entry. Author SummaryCalcium channels represent potential targets to parasitic helminths. We investigated Schistosoma mansoni CRAC channel activation through the expression of its proteins. We have established that the fundamental protein conformational changes and protein-protein interactions underlying STIM-ORAI signaling are shared between humans and schistosome proteins. Importantly, a key finding is that evolutionary divergence in residues that are not implicated in the basic mechanisms of STIM-ORAI activation appears to offer a window for pharmacological inhibitors that would be selective for the schistosome ORAI channel. We identified pharmacological differences for two compounds tested. These differences open avenues for the development of selective drugs that can target the S. mansoni CRAC channel without affecting human physiology, thus offering the prospect of new treatments for schistosomiasis.

Pseudomonas aeruginosa relies on heme acquisition to sustain growth and virulence in the iron-limited environment of the host, particularly in the acidic airways of patients with cystic fibrosis where antibiotic efficacy is markedly reduced. The Has heme assimilation system is initiated by the secreted hemophore HasA, which binds extracellular heme and delivers it to the outer-membrane receptor HasR. During infection, HasA is proteolytically processed, generating a truncated form that constitutes the biologically relevant species. Here, we characterise the structural and functional properties of truncated HasA under disease-associated acidic conditions (pH 6.5) and perform a rationally designed Systemic Evolution of Ligands by EXponential enrichment (SELEX) method to identify DNA aptamers capable of binding this hemophore. Biophysical analyses revealed that truncated HasA is folded, displays a mixed /{beta} secondary structure, and exists as a concentration-dependent mixture of monomers and domain-swapped dimers. Circular dichroism and nano-differential scanning fluorimetry identified two thermal transitions, consistent with the coexistence of apo/holo and monomer/dimer species. The apo protein bound heme with high affinity (KD = 113 nM) and a 1:1 stoichiometry, confirming preservation of its functional binding mechanism. Twelve rounds of SELEX, incorporating target switching and platform switching, yielded a highly enriched aptamer pool dominated by two sequences, HasA_1 and HasA_2. ELONA (Enzyme-Linked Oligonucleotide Assay) assays demonstrated that both aptamers bind specifically to truncated HasA, and biolayer interferometry revealed nanomolar dissociation constants (KD = 0.59 M and 0.32 M, respectively). These aptamers did not bind full-length HasA, BSA, or control sequences, confirming that selection under acidic conditions drove specificity toward the physiologically relevant form of the hemophore. Our findings identify HasA-binding DNA aptamers that retain function in acidic environments where antibiotic potency is compromised, highlighting their potential as molecular tools to disrupt heme acquisition in P. aeruginosa. This work establishes a foundation for developing aptamer-based antimicrobial strategies targeting the Has system, particularly under acidic conditions where conventional antibiotics are compromised.

Cholera is a diarrheal disease from colonization of the small intestine by the Gram-negative bacterium Vibrio cholerae. Cholera remains a critical public health concern due to limitations in therapy and rising antibiotic resistance. The peptide-binding domain (PBD) of the adhesion protein FrhA is critical for V. cholerae intestinal colonization and biofilm formation. In a program targeted on blocking cholera infection, antagonists of the FrhA-PBD are described based on a tripeptide motif. Notably, constraint of the N-terminal tryptophan residue using 1,2,3,4-tetrahydro-{beta}-carboline-3-carboxylic acid (Tcc) and co-crystallization of H-(S)-Tcc-Thr-Asp-OH with an FrhA construct has provided structural information to guide inhibitor design. The diastereomer (R)-Tcc-Thr-Asp exhibited nanomolar binding affinity (Kd = 101 {+/-} 30.2 nM). H-(R)-Tcc-Thr-Asp-OH blocked bacterial hemagglutination mediated by FrhA-PBD and reduced biofilm formation in vitro. Moreover, it showed greater resistance to the digestive enzyme chymotrypsin than previously reported inhibitory pentapeptides, offering potential proteolytic stability while blocking FrhA-PBD-mediated adhesion in the intestine.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

SH2 domains are phosphotyrosine-binding modules that play a critical role in cell signaling by mediating protein-protein interactions. While tyrosine phosphorylation has been shown to impact SH2 domain function in signaling, the specific effects of phosphorylation at different sites within the domain remain poorly understood. In this study, we selected two conserved regions of tyrosine phosphorylation within SH2 domains, near conserved binding interface residues, and developed approaches to evaluate the impact of those sites on ligand binding. Using a modified dot blot assay to screen phosphomimic mutations, we studied specific tyrosine residues within the PTPN11-N, LYN, and SYK-C SH2 domains, finding that the PTPN11 N-terminal site (Y63) modulates the specificity, reducing binding of physiologically relevant substrates. Our findings provide new insights into the regulatory mechanisms governing SH2 domain function and highlight the importance of site-specific phosphorylation in modulating protein-protein interactions in cell signaling pathways.

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

Sorcin is a penta-EF-hand Ca2+-binding protein that acts as a Ca2+ sensor and regulator of Ca2+ homeostasis. Although the structure and Ca2+-dependent activation of Sorcin is well characterized, the function of its flexible N-terminal domain (NTD) remains unclear. We combined sequence analysis, Ca2+-induced aggregation assays, multidimensional NMR spectroscopy, and long-timescale molecular dynamics (MD) simulations to define the NTDs role in Sorcin activation. Sequence comparisons showed that the NTD is conserved across vertebrates despite its intrinsic disorder, indicating functional importance. NTD deletion markedly reduced Ca2+ responsiveness. Relative to full-length Sorcin, the construct with NTD truncation required more than twofold higher Ca2+ concentrations and over tenfold higher protein concentrations to initiate aggregation, while aggregation kinetics slowed by nearly three orders of magnitude. Temperature-dependent measurements yielded an apparent activation energy of 36.7 kJ{middle dot}mol-1, consistent with aggregation driven by Ca2+-induced conformational activation rather than denaturation. NMR chemical shift perturbations localized the effects of NTD removal to the EF-hand Ca2+-binding loops and adjacent D-helix. Apo-state MD simulations revealed transient intra- and intermolecular NTD-SCBD contacts that explain some perturbations and support direct and allosteric regulation. Ca2+-bound simulations further showed that SCBD departs more readily from the crystallographic active conformation than full-length Sorcin, indicating that the NTD stabilizes the active state through dynamic contacts. Together, these findings establish the NTD as a critical regulator that enhances Ca2+ responsiveness by shifting Sorcins conformational equilibrium toward the active state and suggest that flexible N-terminal extensions regulate signaling within the penta-EF-hand protein family.

The participation of sulfhydryl or thiol functions in a multitude of protein posttranslational modifications, although reflects on the redox versatility of cysteine residues, but their assessment in a dynamic cellular milieu involving the facile inter-conversion of SH to SSG, S-S, SNO, and S-R has been overwhelmingly difficult despite theirimplications in protein folding, enzyme structure and function, signalling and detoxification pathways, and pathophysiological ramifications.The current methodology, in contrast to a wide variety of cumbersome and prolonged techniques,repurposes the conventional DTNB assay for a hassle-free qualitative and quantitative analysisof redox-modified single or multiple susceptible thiol residues of cysteines in pure proteins as well as in a complex mixture of proteins.In this study, we document the thiol content, bearing the susceptibility to undergo reversible, oxidative thiol modifications, utilizing reverse DTNB assay in cell-free lysates and purified proteins that might provide a possible framework for dissecting the physiological phenomena behind the concealment of the susceptible cysteines through their redox-modified forms.