Journal of Biological Chemistry

Matrix metalloproteinase 13 (MMP13) is a zinc-dependent protease that plays key roles in extracellular matrix remodeling. Like several other MMPs, MMP13 has been shown to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan found at the cell surface and in the extracellular matrix, but the significance of the interaction remains unknown. Here we report that while zymogen and mature forms of MMP13 both bind HS with high affinity, their interactions with HS display markedly different characteristics in terms of preferred HS structure and binding kinetics. By structure-guided mutagenesis, we identified a large HS-binding site of MMP13 consists of 10 residues in the hemopexin domain, 3 residues in the catalytic domain, and 2 residues in the linker region. While these basic residues participate in binding to both zymogen and mature forms of MMP13, the relative contribution of many residues differs substantially between the two forms, which likely contributes to their distinct HS-binding characteristics. Binding of HS to mature MMP13 resulted in selective inhibition of the collagenase activity of MMP13 in a length- and sulfation-dependent manner, but the binding had no effect on degradation of non-collagen substrates. Mechanistically, the inhibitory effect of HS likely results from reduced interdomain flexibility after binding of HS, and/or HS-induced dimerization of MMP13. In sum, our study establishes HS as a multifaceted regulator of MMP13 activity, and discovers that the HS-binding site of MMP13 is a novel exosite that can be targeted to inhibits its collagenase activity.

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

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.

The cofactor Fe-protoporphyrin IX cofactor (heme) performs many functions in biology. Animal cells must stabilize their newly generated heme-free (apo)-hemeproteins and deliver mitochondrial heme to them so they can mature to functional form. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) typically accomplishes the heme deliveries, and for many apo-hemeproteins, heat shock protein 90 (Hsp90) drives their heme insertions. We previously observed hemeproteins express poorly in a cell line (COS-7) that does not express soluble guanylyl cyclase (sGC), a heme-binding enzyme that typically functions through its cGMP generation. To understand sGC involvement, we expressed four hemeproteins, Hemoglobin beta (Hb{beta}), Myoglobin (Mb), Indoleamine 2,3-dioxygenase 1 (IDO1), and Tryptophan 2,3-dioxygenase (TDO) in a cell line expressing sGC (HEK293) or in two cell lines (COS-7, DU145) that do not. We assessed hemeprotein expression levels, their abilities to acquire heme, and when relevant if these facets could be rescued by co-expressing individual sGC subunits, including variants with defects in either sGC heme binding, Hsp90 association, heterodimerization, or cGMP production. We found that co-expression of either sGC subunit was essential for three of the four apo-hemeproteins to accumulate in the COS7 and DU145 cells and acquire heme. This did not involve heme binding, heterodimer formation, or cGMP generation by the sGC subunits, and instead depended on a subunits ability to recruit Hsp90 and GAPDH to the apo-hemeproteins via their own Hsp90 binding. Recruiting Hsp90 and GAPDH to apo-hemeprotein clients to ensure they can accumulate and mature to functional form broadens our understanding of sGC and Hsp90 functions in biology.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.

The cluster of differentiation 36 (CD36) is a membrane protein with broad physiological roles in health and disease, and its function is regulated in part by phosphorylation. Experimental evidence shows that phosphorylation of Thr92 reduces CD36 affinity for thrombospondin-1 (TSP-1), binding of which initiates antiangiogenic signaling, whereas phosphorylation of Ser237 decreases CD36-mediated fatty acid uptake, with implications for energy metabolism. However, the only available crystal structure of CD36 lacks phosphorylation, and the molecular mechanisms by which phosphorylation regulates CD36 function remain largely unknown. This study provides an atomically detailed computational characterization of CD36 in unphosphorylated and dual phosphorylated states, using molecular dynamics simulations with a total sampling time of 30 microseconds in combination with Markov state models. We present, to our knowledge, the first evidence of a cryptic pocket on CD36 surface that is formed by phosphorylation. This cryptic surface pocket and a loop spanning residues 121-131 form a high affinity binding site for TSP-1 derived ligands, shifting their binding away from the canonical site. We propose that this altered binding provides a molecular basis for the disruption of antiangiogenic signaling upon CD36 phosphorylation. Additionally, our data indicate that, phosphorylation increases helicity and compaction within the helix-loop region spanning residues 296-331, narrowing one of the entrances to the internal cavity and reducing its overall volume. These conformational changes provide a potential mechanistic explanation for the decrease in fatty acid uptake upon CD36 phosphorylation. Our findings provide structural insights that may inform the future design of CD36 modulators and emphasize the importance of targeting phosphorylation induced CD36 conformations in angiogenic and metabolic diseases.

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

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.

Mosaic penA alleles encoding highly mutated variants of penicillin-binding protein 2 (PBP2) are the principal determinants of ceftriaxone resistance in Neisseria gonorrhoeae. Resistance-associated mutations in PBP2 from the ceftriaxone-resistant strain H041 restrict formation of the inward conformation of the {beta}3-{beta}4 loop associated with efficient acylation, but how {beta}-lactam recognition is coupled to this conformational switch is unknown. Because the conserved active-site residue Tyr422 interacts with the R1 substituent of {beta}-lactams, we investigated its role in coupling ligand recognition and acylation activity. Mutation of Tyr422 to Ala lowered acylation rates by up to 120-fold for cefoperazone and piperacillin, whereas acylation rates of ceftriaxone increased 4-fold. Unexpectedly, the crystal structure of the Y422A mutant acylated by ceftriaxone revealed that the {beta}3-{beta}4 loop had adopted the inward, high-activity conformation, despite position 422 being spatially distant from the loop. Transformation experiments showed that cell viability requires a tyrosine at position 422, indicating the residue is essential for transpeptidase function. Together, these findings reveal an energetic coupling between an active-site residue in PBP2 and a conformational switch whose equilibrium is altered by resistance mutations. The previously observed higher activity of {beta}-lactams containing extended R1 groups is consistent with stronger interactions with Tyr422 that favor the conformational switch. Molecular modeling suggests that such groups enhance activity by mimicking the iso-Glu region of the pentapeptide substrate. Overall, we propose that access to the high-activity state of PBP2 where the {beta}3-{beta}4 loop is inward is regulated by interactions between Tyr422 and {beta}-lactam R1 groups, and that resistance mutations function by tilting the balance toward a lower activity state.

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

Sliding clamp proteins play central roles in DNA metabolism, including replication and repair. The ring-shaped E. coli beta clamp accommodates double-stranded DNA and serves as a platform for proteins involved in multiple DNA transactions. The inner pore of the beta clamp harbors a series of positively charged and polar residues that can bind to the negatively charged backbone of the DNA. These residues are arrayed so that they do not align with the charged phosphates of the DNA backbone. It is hypothesized that this arrangement of these residues provides for the movement of the clamp on DNA as it alternates which residues are bound to the DNA backbone. In this work, we mutated specific charged and polar residues that project into the inner pore of the beta clamp. The beta clamp variants are dimers and have similar thermal stability and in general a similar ability to complement a temperature sensitive strain for growth. One exception was beta-Q149A, which appeared as higher-order species on a native gel although its hydrogen-deuterium exchange pattern measured by mass spectrometry was overall similar to WT beta. These variants all had decreased binding to DNA after loading. Optical tweezers experiments were used to monitor loading on single DNA molecules and measure the rate of beta clamp sliding on DNA. Consistent with the hypothesized role of positively charged residues in the beta inner pore, mutation of one residue resulted in a faster rate of sliding on DNA.

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.

Biomolecular condensates are membraneless compartments critical for the functional organization of cellular macromolecules in essential processes such as cell division, gene transcription or stress responses. We previously reported that vimentin filaments remodel into phase separated biomolecular condensates upon oxidative stress. This process requires vimentin single cysteine, C328, suggesting the involvement of oxidative modifications of this residue. Here, we aimed to generate vimentin condensates by inserting mutations mimicking posttranslational modifications associated with oxidative stress. In vimentin deficient cells, a cysteine oxidation mimetic mutant, vimentin C328D, formed only elongated particles or short filaments that evolved towards droplets upon serum deprivation or treatment with the oxidant diamide. Among vimentin posttranslational modifications rapidly responding to these stimuli, glycosylation confers filament stability whereas phosphorylation promotes disassembly. We observed that the O-deglycosylation inhibitor thiamet G, and the kinase inhibitors staurosporine and H-89, attenuated diamide-elicited vimentin C328D droplet formation, suggesting a potential glycosylation/phosphorylation interplay in this effect. Indeed, introducing phosphomimetic residues at certain single vimentin glycosylation and/or phosphorylation sites induced the formation of droplets, only if combined with the C328D mutation. In particular, the vimentin S49D,C328D mutant formed condensates that were reversibly dispersed by dilution through hypotonic shock. Therefore, mimicking C328 oxidation and S49 phosphorylation was sufficient to elicit vimentin phase separation. In vitro, purified vimentin S49D,C328D polymerized into a mixture of aberrant filaments and aggregates, which, in the presence of crowders, evolved towards paracrystals or clusters of beaded assemblies depending on pH. These findings highlight the role of C328 perturbations in the formation of biomolecular condensates and suggest a modulatory role of glycosylation/phosphorylation, thus shedding light on the processes regulating vimentin phase separation.

Syndecans are transmembrane proteoglycans that regulate angiogenesis through both their glycosaminoglycan chains and core proteins. While roles for all four mammalian syndecans in new blood vessel formation are well established, it has more recently emerged that their extracellular core proteins contain discrete bioactive regulatory sequences capable of influencing cellular processes, including angiogenesis. We previously demonstrated that the syndecan-3 (SDC3) ectodomain possesses anti-angiogenic activity independent of its heparan sulphate chains. Here, we identified and characterised a novel anti-angiogenic sequence within the SDC3 ectodomain. Using recombinant truncation mutants, endothelial migration assays and peptide mapping, we localised activity to a discrete region of the extracellular domain and subsequently defined a conserved minimal nine amino acid peptide, QM111, that retained full biological activity. QM111 inhibited endothelial cell migration and angiogenic sprouting in both rat aortic ring and mouse choroidal explant models. Intrinsic disorder analysis revealed that QM111 resides within a region of comparatively reduced disorder, consistent with other syndecan regulatory sequences. This supports the concept that syndecan ectodomains contain conserved functional modules embedded within intrinsically disordered extracellular domains. QM111 did not induce inflammatory chemokine production, exhibited no detectable cytotoxicity, and retained substantial stability in human serum and vitreous humour. Finally, QM111 displayed anti-angiogenic activity comparable to the previously described syndecan-2-derived peptide QM107, with combination treatment producing more robust inhibition of angiogenesis. These findings identify QM111 as a novel endogenous anti-angiogenic peptide and support the concept that syndecan ectodomains are reservoirs of biologically active regulatory sequences with therapeutic potential. The work further establishes syndecan-derived peptides as a promising platform for the development of next-generation anti-angiogenic therapies.

AMP-activated protein kinase (AMPK) regulates metabolism in response to metabolic stress that includes stimulating glucose uptake in skeletal muscle independently of the canonical insulin signalling pathway, positioning it as an attractive therapeutic target for insulin resistance and type 2 diabetes mellitus (T2DM). AMPK is an {beta}{gamma} heterotrimer, with multiple isoforms for each subunit enabling the formation of 12 different complexes with distinct tissue expression profiles. Among these, the 2{beta}2{gamma}3 complex is predominantly expressed in skeletal muscle, the major site of glucose disposal and a highly desirable therapeutic target for T2DM. Here, we characterise the functional role of a unique, 182 residue N-terminal extension (NTE) within {gamma}3 subunit. Deletion of the {gamma}3-NTE from 2{beta}2{gamma}3 complex increases basal AMPK activity without affecting activation by AMP or pharmacological AMPK activators, demonstrating the {gamma}3-NTE performs an autoinhibitory function. Using complementary biophysical techniques, including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, chemical crosslinking and co-pulldowns, we identified a 39-residue sequence in the {gamma}3-NTE (residues 129-168), that directly interacts with the C-helix of the AMPK kinase domain small lobe, a key regulatory element in many protein kinases. Using AlphaFold3, we probe the interaction predicted to take place between a {gamma}3-NTE -helix ({gamma}3-iHelix; [~]T142-E154) and the C-helix in the 2{beta}2{gamma}3 complex. These findings provide the groundwork for developing novel T2DM therapies that target AMPK activation selectively in skeletal muscle involving reversal of the {gamma}3 autoinhibition.

Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.

The HIV-1 accessory protein Nef plays a central role in viral pathogenesis by enhancing viral replication, modulating cellular signaling, and evading immune recognition, making it a compelling target for therapeutic intervention. Nef lacks enzymatic activity and instead functions through diverse interactions with host cell proteins including the Src-family tyrosine kinase, Hck. Kinase activation may requires Nef homodimerization, as mutations disrupting the Nef dimer interface impair kinase activation as well as many other Nef functions. In the present study, we investigated the structural consequences of dimer interface mutations and their impact on Nef interactions with Hck regulatory domains. Using size-exclusion chromatography, multi-angle light scattering and crystallography, we found that mutations at dimer interface residues Leu112 and Phe121 abolish recombinant Nef protein dimerization while preserving the overall Nef fold, resulting in monomeric 1:1 complexes with Hck SH3 or SH3-SH2 domain proteins. These findings demonstrate that the broad phenotypic effects of interface mutations arise from loss of Nef dimerization rather than global misfolding or perturbation of SH3 binding. We also investigated the effects of small molecule Nef inhibitors on homodimer formation. These compounds, like the dimerization-defective mutations, suppress kinase activation, viral replication and restore immune recognition of HIV-infected cells. Using a SplitFAST fluorescence complementation assay, we provide direct evidence that these inhibitors disrupt Nef homodimer formation in solution. Co-crystallization of a wild-type Nef:SH3 complex with an inhibitor also prevented homodimer formation. Computational docking identified a shared pocket for six active Nef inhibitors formed by the Nef dimer interface but lost in the monomer. Together, our findings support homodimerization as a structural feature essential for many Nef functions and validate disruption of this interface as a promising therapeutic strategy against HIV-1.