Substrate Recognition Mechanism Research Articles

Mechanistic Diversity and Functional Roles Define the Substrate Specificity and Ligand Binding of Bacterial PGP Phosphatases

Phosphatidylglycerol (PG) is a critical membrane phospholipid in microorganisms, synthesized via the dephosphorylation of phosphatidylglycerol-phosphate (PGP) by three membrane-bound phosphatases: PgpA, PgpB, and PgpC. While any one of these enzymes can produce PG at wild-type levels, the reason for the presence of all three in bacteria remains unclear. To address this question, we characterized these phosphatases in vitro to uncover their mechanistic differences. Our assays demonstrated that all three enzymes catalyze the hydrolysis of PGP but exhibit distinct substrate selectivity. PgpB displays a broad substrate range, dephosphorylating various lipid phosphates, while PgpA and PgpC show a higher specificity for lysophosphatidic acid and PGP. Notably, PgpA also effectively dephosphorylates soluble metabolites, such as glycerol-3-phosphate and glyceraldehyde-3-phosphate, suggesting its unique substrate-binding mechanism that relies on precise recognition of the glycerol head group rather than the fatty acid. Inhibitor screening with synthetic substrate analogs revealed that PgpB is inhibited by lipid-like compounds XY-14 and XY-55, whereas PgpA and PgpC are unaffected. Structural analysis and mutational studies identified two charged residues at the catalytic site entry for inhibitor binding in PgpB and support the notion that the PgpB maintains a large substrate binding site to accommodate multiple ligand binding conformations. These findings underscore the distinct substrate recognition mechanisms and possible functional roles of PgpA, PgpB, and PgpC in bacterial lipid metabolism and offer insights for developing novel inhibitors targeting bacterial membrane phospholipid biosynthesis.

The β-Ketoacyl-ACP Synthase FabF Catalyzes Carbon-Carbon Bond Formation in a Bimodal Pattern for Fatty Acid Biosynthesis.

Fatty acids produced by the type-II fatty acid biosynthetic pathway (FAS-II) are essential biomaterials for bacterial membrane construction and numerous metabolic routes. The β-ketoacyl-ACP synthase FabF catalyzes the key C-C bond formation step for fatty acid elongation in FAS-II. Here, we revealed the substrate recognition and catalytic mechanisms of FabF by determining FabF-ACP complexes. FabF displays a distinctive bimodal catalytic pattern specifically on C6 and C10 acyl-ACP substrates. It utilizes positively charged residues located on the η3-helix and loop1 regions near the catalytic tunnel entrance to bind ACP, and two hydrophobic cavities as well as "front", "middle", and "back" door residues to specifically stabilize C6 and C10 acyl substrates for preferential catalysis. Further quantum chemistry calculations suggest that the FabF catalytic residues Lys336 and His304 facilitate proton transfer during condensation catalysis and C-C bond formation. Our results provide key mechanistic insights into the biosynthesis of molecular carbon skeletons based on ketosynthases that are highly conserved through the FAS and polyketide synthase (PKS) analogous biosynthetic routes, broaden the understanding of the tricarboxylic acid cycle that utilizes lipoic acid derived from C8-ACP accumulated due to the FabF distinctive catalytic pattern for oxidative decarboxylations, and may facilitate the development of narrow-spectrum antibacterial drugs.

The β‐Ketoacyl‐ACP Synthase FabF Catalyzes Carbon‐Carbon Bond Formation in a Bimodal Pattern for Fatty Acid Biosynthesis

AbstractFatty acids produced by the type‐II fatty acid biosynthetic pathway (FAS‐II) are essential biomaterials for bacterial membrane construction and numerous metabolic routes. The β‐ketoacyl‐ACP synthase FabF catalyzes the key C−C bond formation step for fatty acid elongation in FAS‐II. Here, we revealed the substrate recognition and catalytic mechanisms of FabF by determining FabF‐ACP complexes. FabF displays a distinctive bimodal catalytic pattern specifically on C6 and C10 acyl‐ACP substrates. It utilizes positively charged residues located on the η3‐helix and loop1 regions near the catalytic tunnel entrance to bind ACP, and two hydrophobic cavities as well as “front”, “middle”, and “back” door residues to specifically stabilize C6 and C10 acyl substrates for preferential catalysis. Further quantum chemistry calculations suggest that the FabF catalytic residues Lys336 and His304 facilitate proton transfer during condensation catalysis and C−C bond formation. Our results provide key mechanistic insights into the biosynthesis of molecular carbon skeletons based on ketosynthases that are highly conserved through the FAS and polyketide synthase (PKS) analogous biosynthetic routes, broaden the understanding of the tricarboxylic acid cycle that utilizes lipoic acid derived from C8‐ACP accumulated due to the FabF distinctive catalytic pattern for oxidative decarboxylations, and may facilitate the development of narrow‐spectrum antibacterial drugs.

Structural insights into the membrane-bound proteolytic machinery of bacterial protein quality control.

In bacteria and eukaryotic organelles of prokaryotic origin, ATP-dependent proteases are crucial for regulating protein quality control through substrate unfolding and degradation. Understanding the mechanism and regulation of this key cellular process could prove instrumental in developing therapeutic strategies. Very recently, cryo-electron microscopy structural studies have shed light on the functioning of AAA+ proteases, including membrane-bound proteolytic complexes. This review summarizes the structure and function relationship of bacterial AAA+ proteases, with a special focus on the sole membrane-bound AAA+ protease in Escherichia coli, FtsH. FtsH substrates include both soluble cytoplasmic and membrane-incorporated proteins, highlighting its intricate substrate recognition and processing mechanisms. Notably, 12 copies of regulatory HflK and HflC proteins, arranged in a cage-like structure embedded in the bacterial inner membrane, can encase up to 4 FtsH hexamers, thereby regulating their role in membrane protein quality control. FtsH represents an intriguing example, highlighting both its similarity to cytosolic AAA+ proteases with respect to overall architecture and oligomerization as well as its unique features, foremost its incorporation into a membrane-bound complex formed by HflK and HflC to mediate its function in protein quality control.

Interleukin-2 receptor α (IL-2Rα/CD25) shedding is differentially regulated by N- and O-glycosylation

The cytokine interleukin-2 (IL-2) is a critical regulator of immune responses, with an especially well-characterized role in regulating T-cell homeostasis. IL-2 signaling involves three distinct receptor subunits: the IL-2Rα (CD25), IL-2Rβ, and IL-2Rγ. The intracellular transduction of IL-2-induced signals is strictly dependent on IL-2Rβ and IL-2Rγ, while the IL-2Rα is not directly involved in signaling. Instead, it has the highest affinity towards IL-2 and is thus responsible for regulating the affinity of a cell for IL-2. In addition to the membrane-bound IL-2Rα, a soluble form of the receptor (sIL-2Rα) has been described, which is present in the blood of healthy individuals, increased under various pathological conditions, and able to bind IL-2 and thus modulate its function. The sIL-2Rα is generated by proteolytic cleavage of the membrane-bound receptor. Here, we analyze whether glycosylation of the IL-2Rα regulates its proteolysis. We find that constitutive IL-2Rα shedding is affected by glycosylation and discover distinct roles for N- and O-glycosylation. Furthermore, we show that induced shedding by the metalloproteases ADAM10 and ADAM17 is also differentially regulated by distinct types of glycans. Finally, we identify a specific role for an N-glycan at an exosite in ADAM17-mediated proteolysis that does not affect ADAM10, indicating distinct substrate recognition mechanisms. These results further the understanding of the mechanisms leading to sIL-2Rα generation, and thus offer the opportunity to specifically modulate the generation of the soluble receptor.

The importance of bacteria of the genus Bifidobacterium in intestinal microbiocenosis

Bifidobacteria are the most common microorganisms in the intestines of healthy breastfed children. The genus Bifidobacterium includes bacteria characterized by probiotic properties, such as the induction of immunomodulators, increasing the nutritional value of products due to the assimilation of substrates that are not broken down by the host, anticarcinogenic activity, synthesis of vitamins, production of antimicrobial drugs, which contribute to the promotion of health. Bifidobacteria demonstrate physiological and genetic characteristics including adhesion to the intestinal epithelium, as well as metabolism of glycans in the host body. Multitrophic interaction is formed based on various mechanisms of substrate recognition in the surrounding environment and the transmission of molecular and genetic information, which contributes to survival in the human gastrointestinal tract. Representatives of the Bifidobacterium bifidum species constitute a dominant taxon among bifidobacteria, demonstrating significant probiotic properties and extensive potential for the treatment and prevention of various diseases. Currently, a large number of Bifidobacterium bifidum species have been sequenced, which are of interest to medicine, biotechnology, and agriculture. Their genetic strategies for colonizing and persisting in the human intestine have been identified. Cross-interaction mechanisms of Bifidobacterium bifidum with the host and other microorganisms have been demonstrated using various structures. In this review, we discuss current knowledge about the biology of the genus Bifidobacterium, including the biological characteristics of Bifidobacterium bifidum species, which exhibit specific adaptations to the human intestine.

Kinetic mechanism and determinants of EF-P recruitment to translating ribosomes.

EF-P is a translation factor that facilitates the formation of peptide bonds between consecutive prolines. Using FRET between EF-P and ribosomal protein bL33, we studied dynamics and specificity of EF-P binding to the ribosome. Our findings reveal that EF-P rapidly scans for a free E site and can bind to any ribosome containing a P-site tRNA, regardless of the ribosome's functional state. The interaction with uL1 is essential for EF-P binding, while the β-Lys modification of EF-P doubles the association rate. Specific interactions with the D-loop of tRNAPro or tRNAfMet and via the β-Lys group with the tRNA in the peptidyl transferase center reduce the rate of EF-P dissociation from the ribosome, providing the specificity for complexes that need help in catalyzing peptide bond formation. The nature of the E-site codon has little effect on EF-P binding kinetics. Although EF-P dissociation is reduced upon recognizing its correct tRNA substrate, it remains sufficiently rapid compared to tRNA translocation and does not affect the translocation rate. These results highlight the importance of EF-P's scanning-engagement mechanism for dynamic substrate recognition during rapid translation.

Structural Insights into the Substrate Recognition and Catalytic Mechanism of a GH16 βκ-Carrageenase from Wenyingzhuangia fucanilytica.

Understanding the substrate specificity of carrageenases has long been of interest in biotechnology applications. So far, the structural basis of the βκ-carrageenase that hydrolyzes furcellaran, a major hybrid carrageenan, remains unclear. Here, the crystal structure of Cgbk16A_Wf, as a representative of the βκ-carrageenase from GH16_13, was determined, and the structural characteristics of this subfamily were elucidated for the first time. The substrate binding mode was clarified through a structure analysis of the hexasaccharide-bound complex and molecular docking. The binding pocket involves a conserved catalytic motif and several specific residues associated with substrate recognition. Functions of residues R88, E290, and E184 were validated through site-directed mutagenesis. Comparing βκ-carrageenase with κ-carrageenase, we proposed that their different substrate specificities are partly due to the distinct conformations of subsite -1. This research offers a comprehensive understanding of the recognition mechanism of carrageenases and provides valuable theoretical support for enzyme modification and carrageenan oligosaccharide preparation.

Substrate binding and inhibition mechanism of norepinephrine transporter.

Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level1. Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target1. However, the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states-the apo state, and in states bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed extracellular gate and an open intracellular gate. The substrate MIBG binds at the centre of hNET. Radafaxine also occupies the substrate-binding site and might block the structural transition of hNET for inhibition. These structures provide insights into the mechanism of substrate recognition and orthosteric inhibition of hNET.

Structural insight into binding site access and ligand recognition by human ABCB1.

ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, its mechanisms of poly-specific substrate recognition and transport remain poorly resolved. Here we present cryo-EM structures of lipid embedded human ABCB1 in its apo, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4-3.9 Å resolution without using stabilizing antibodies or mutations and each revealing a distinct conformation. The substrate binding site is located within one half of the molecule and, in the apo state, is obstructed by transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major differences in TM arrangement and ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Our data offer fundamental new insights into the role structural asymmetry, secondary structure breaks, and lipid interactions play in ABCB1 function and have far-reaching implications for ABCB1 inhibitor design and predicting its substrate binding profiles.

Molecular basis of human noradrenaline transporter reuptake and inhibition.

Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types1. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter (NET) located in the presynaptic membrane2. Here we report the cryo-electron microscopy (cryo-EM) structures of the human NET in both its apo state and when bound to substrates or antidepressant drugs, with resolutions ranging from 2.5 Å to 3.5 Å. The two substrates, noradrenaline and dopamine, display a similar binding mode within the central substrate binding site (S1) and within a newly identified extracellular allosteric site (S2). Four distinct antidepressants, namely, atomoxetine, desipramine, bupropion and escitalopram, occupy the S1 site to obstruct substrate transport in distinct conformations. Moreover, a potassium ion was observed within sodium-binding site 1 in the structure of the NET bound to desipramine under the KCl condition. Complemented by structural-guided biochemical analyses, our studies reveal the mechanism of substrate recognition, the alternating access of NET, and elucidate the mode of action of the four antidepressants.

Molecular insights into degron recognition by CRL5ASB7 ubiquitin ligase

The ankyrin (ANK) SOCS box (ASB) family, encompassing ASB1–18, is the largest group of substrate receptors of cullin 5 Ring E3 ubiquitin ligase. Nonetheless, the mechanism of substrate recognition by ASB family proteins has remained largely elusive. Here we present the crystal structure of ASB7-Elongin B-Elongin C ternary complex bound to a conserved helical degron. ASB7 employs its ANK3-6 to form an extended groove, effectively interacting with the internal α-helix-degron through a network of side-chain-mediated electrostatic and hydrophobic interactions. Our structural findings, combined with biochemical and cellular analyses, identify the key residues of the degron motif and ASB7 required for their recognition. This will facilitate the identification of additional physiological substrates of ASB7 by providing a defined degron motif for screening. Furthermore, the structural insights provide a basis for the rational design of compounds that can specifically target ASB7 by disrupting its interaction with its cognate degron.

Structural Basis of the Dehydratase Module (hDH) of Human Fatty Acid Synthase.

The human fatty acid synthase (hFASN) produces fatty acids for cellar membrane construction, energy storage, biomolecule modifications and signal transduction. Abnormal expression and functions of hFASN highly associate with numerous human diseases such as obesity, diabetes, and cancers, and thereby it has been considered as a valuable potential drug target. So far, the structural and catalytic mechanisms of most of the hFASN enzymatic modules have been extensively studied, except the key dehydratase module (hDH). Here we presented the enzymatic characterization and the high-resolution crystal structure of hDH. We demonstrated that the hDH preferentially catalyzes the acyl substrates with short lengths between 4 to 8-carbons, and exhibits much lower enzymatic activity on longer substrates. Subsequent structural study showed that hDH displays a pseudo-dimeric organization with a single L-shaped composite hydrophobic catalytic tunnel as well as an atypical ACP binding site nearby, indicating that hDH achieves distinct substrate recognition and dehydration mechanisms compared to the conventional bacterial fatty acid dehydratases identified. Our findings laid the foundation for understanding the biological and pathogenic functions of hFASN, and may facilitate therapeutical drug development against diseases with abnormal functionality of hFASN.

Dimeric transport mechanism of human vitamin C transporter SVCT1

Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5–3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.

Structure and Substrate Recognition of the Escherichia coli Transport Protein NupG from the Nucleoside: H+ Symporter (NHS) Family

The nucleoside transporter NupG is one of the two principal transport proteins in the inner membrane of Escherichia coli that enable the organism to scavenge nucleosides from its external environment. NupG functions in a symport manner driven by the proton motive force and is a member of the Nucleoside:H+ Symporter (NHS) subfamily of the Major Facilitator Superfamily (MFS) of transporters. NupG has broad substrate specificity, transporting all naturally occurring purine and pyrimidine nucleosides. In studies over many years the nupG gene has been cloned and amplified, and the NupG protein has been purified, subjected to biochemical, biophysical and computational analysis, and its X-ray structure determined in the apo state at 3.0 Å resolution. The NupG structure had a typical MFS fold with twelve transmembrane spanning α-helices and distinct N- and C-terminal domains linked by a flexible loop. Preliminary site-directed mutagenesis and molecular docking studies on NupG identified nine putative nucleoside binding pocket residues (R136, T140, F143, Q225, N228, Q261, E264, Y318, F322) and a mutant (D323A) with 20-fold enhanced uridine binding activity. Further biochemical and structural investigations are necessary to better understand the substrate recognition and molecular mechanism of E. coli NHS family proteins (NupG, XapB, YegT).

Intra-chloroplast proteases: A holistic network view of chloroplast proteolysis.

Different proteases and peptidases are present within chloroplasts and nonphotosynthetic plastids to process precursor proteins and to degrade cleaved chloroplast transit peptides and damaged, misfolded, or otherwise unwanted proteins. Collectively, these proteases and peptidases form a proteolysis network, with complementary activities and hierarchies, and build-in redundancies. Furthermore, this network is distributed across the different intra-chloroplast compartments (lumen, thylakoid, stroma, envelope). The challenge is to determine the contributions of each peptidase (system) to this network in chloroplasts and nonphotosynthetic plastids. This will require an understanding of substrate recognition mechanisms, degrons, substrate, and product size limitations, as well as the capacity and degradation kinetics of each protease. Multiple extra-plastidial degradation pathways complement these intra-chloroplast proteases. This review summarizes our current understanding of these intra-chloroplast proteases in Arabidopsis and crop plants with an emphasis on considerations for building a qualitative and quantitative network view.

Molecular mechanism of substrate recognition and cleavage by human γ-secretase.

Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-β (Aβ) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aβ49, Aβ46, and Aβ43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a β-strand that forms a hybrid β-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate β-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new β-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.

Structural basis of chorismate isomerization by Arabidopsis ISOCHORISMATE SYNTHASE1.

Salicylic acid (SA) plays a crucial role in plant defense against biotrophic and semibiotrophic pathogens. In Arabidopsis (Arabidopsis thaliana), isochorismate synthase 1 (AtICS1) is a key enzyme for the pathogen-induced biosynthesis of SA via catalytic conversion of chorismate into isochorismate, an essential precursor for SA synthesis. Despite the extensive knowledge of ICS1-related menaquinone, siderophore, and tryptophan (MST) enzymes in bacteria, the structural mechanisms for substrate binding and catalysis in plant isochorismate synthase (ICS) enzymes are unknown. This study reveals that plant ICS enzymes catalyze the isomerization of chorismate through a magnesium-dependent mechanism, with AtICS1 exhibiting the most substantial catalytic activity. Additionally, we present high-resolution crystal structures of apo AtICS1 and its complex with chorismate, offering detailed insights into the mechanisms of substrate recognition and catalysis. Importantly, our investigation indicates the existence of a potential substrate entrance channel and a gating mechanism regulating substrate into the catalytic site. Structural comparisons of AtICS1 with MST enzymes suggest a shared structural framework with conserved gating and catalytic mechanisms. This work provides valuable insights into the structural and regulatory mechanisms governing substrate delivery and catalysis in AtICS1, as well as other plant ICS enzymes.

New insights into the substrate recognition and transport mechanism of the human sodium-taurocholate cotransporter

New insights into the substrate recognition and transport mechanism of the human sodium-taurocholate cotransporter

Cryo-EM structures of the human NaS1 and NaDC1 transporters revealed the elevator transport and allosteric regulation mechanism.

The solute carrier 13 (SLC13) family comprises electrogenic sodium ion-coupled anion cotransporters, segregating into sodium ion-sulfate cotransporters (NaSs) and sodium ion-di- and-tricarboxylate cotransporters (NaDCs). NaS1 and NaDC1 regulate sulfate homeostasis and oxidative metabolism, respectively. NaS1 deficiency affects murine growth and fertility, while NaDC1 affects urinary citrate and calcium nephrolithiasis. Despite their importance, the mechanisms of substrate recognition and transport remain insufficiently characterized. In this study, we determined the cryo-electron microscopy structures of human NaS1, capturing inward-facing and combined inward-facing/outward-facing conformations within a dimer both in apo and sulfate-bound states. In addition, we elucidated NaDC1's outward-facing conformation, encompassing apo, citrate-bound, and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibitor-bound states. Structural scrutiny illuminates a detailed elevator mechanism driving conformational changes. Notably, the ACA inhibitor unexpectedly binds primarily anchored by transmembrane 2 (TM2), Loop 10, TM11, and TM6a proximate to the cytosolic membrane. Our findings provide crucial insights into SLC13 transport mechanisms, paving the way for future drug design.