Substrate Binding Mechanism Research Articles

Structural insights into the molecular mechanisms of substrate recognition and hydrolysis by feruloyl esterase from Aspergillus sydowii

The depolymerization of lignocellulosic biomass is facilitated by feruloyl esterases (FAEs), which hydrolyze ester bonds between lignin and polysaccharides. Fungal FAEs belonging to subfamily (SF) 6 release precursors such as ferulic acid derivatives, attractive for biochemical production. Among these, Aspergillus sydowii FAE (AsFaeE), an SF6 FAE, exhibits remarkable activity across various substrates. In this study, we conducted X-ray crystallography and kinetic analysis to unravel the molecular mechanisms governing substrate recognition and catalysis by AsFaeE. AsFaeE exhibits a typical α/β-hydrolase fold, characterized by a catalytic triad of serine, aspartate, and histidine. Comparative analysis of substrate-free, ferulic acid-bound, and sinapic acid-bound forms of AsFaeE suggests a conformational change in the loop covering the substrate-binding pocket upon binding. Notably, Pro158 and Phe159 within this loop cover the phenolic part of the substrate, forming three layers of planar rings. Our structure-based functional mutagenesis clarifies the roles of the residues involved in substrate binding and catalytic activity. Furthermore, distinct substrate-binding mechanisms between AsFaeE and other studied FAEs are identified. This investigation offers the initial structural insights into substrate recognition by SF6 FAEs, equipping us with structural knowledge that might facilitate the design of FAE variants capable of efficiently processing a wider range of substrate sizes.

In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily (MFS) transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under four ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable super-complex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.

New structures of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis provide a framework for a novel catalytic mechanism for the entire class.

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the 〈-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.

Crystal structure of phosphate bound Acyl phosphatase mini-enzyme from Deinococcus radiodurans at 1Å resolution

Acylphosphatase (Acp) is a hydrolase which specifically cleaves carboxyl-phosphate bond of intermediates of metabolic pathways. It is a small cytosolic enzyme found in both prokaryotic and eukaryotic organisms. Previous crystal structures of acylphosphatase from different organisms have provided insights into the active site but the complete understanding of substrate binding and catalytic mechanisms in acylphosphatase remain elusive. Here we report the crystal structure of phosphate bound acylphosphatase from a mesothermic bacterium, Deinococcus radiodurans (drAcp) at resolution of 1.0 Å. Our structural analysis shows how the terminal phosphate group of substrates is bound to the active site, highlighting the importance of arginine in substrate recognition, role of asparagine in mode of catalysis and shedding light on the reaction mechanism. Additionally, the protein can refold after thermal melting by gradually lowering the temperature. To further explore the dynamics of drAcp, molecular dynamics simulation of drAcp and homologs from thermophilic organisms were carried out which revealed similar root mean square fluctuation profile but drAcp showed comparatively higher fluctuations.

Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

Rational Design of Daunorubicin C-14 Hydroxylase Based on the Understanding of Its Substrate-Binding Mechanism.

Doxorubicin is one of the most widely used antitumor drugs and is currently produced via the chemical conversion method, which suffers from high production costs, complex product separation processes, and serious environmental pollution. Biocatalysis is considered a more efficient and environment-friendly method for drug production. The cytochrome daunorubicin C-14 hydroxylase (DoxA) is the essential enzyme catalyzing the conversion of daunorubicin to doxorubicin. Herein, the DoxA from Streptomyces peucetius subsp. caesius ATCC 27952 was expressed in Escherichia coli, and the rational design strategy was further applied to improve the enzyme activity. Eight amino acid residues were identified as the key sites via molecular docking. Using a constructed screening library, we obtained the mutant DoxA(P88Y) with a more rational protein conformation, and a 56% increase in bioconversion efficiency was achieved by the mutant compared to the wild-type DoxA. Molecular dynamics simulation was applied to understand the relationship between the enzyme's structural property and its substrate-binding efficiency. It was demonstrated that the mutant DoxA(P88Y) formed a new hydrophobic interaction with the substrate daunorubicin, which might have enhanced the binding stability and thus improved the catalytic activity. Our work lays a foundation for further exploration of DoxA and facilitates the industrial process of bio-production of doxorubicin.

Crystal Structure and Functional Characterization of an S-Formylglutathione Hydrolase (BuSFGH) from Burkholderiaceae sp.

S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of Burkholderiaceae sp. SFGH (BuSFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of BuSFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that BuSFGH formed a stable dimer in solution. The enzyme activity assay indicated that BuSFGH has a high preference for short-chain p-nitrophenyl esters, such as p-nitrophenyl acetate. The activity of BuSFGH toward p-nitrophenyl acetate was five times higher than that of p-nitrophenyl butylate. Molecular modeling studies on the p-nitrophenyl acetate-bound BuSFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of BuSFGH. They can also provide useful insights for designing modified BuSFGHs with different substrate specificities.

Structural Basis for Substrate Binding and Catalytic Mechanism of the Key Enzyme VioD in the Violacein Synthesis Pathway.

Violacein is a pigment synthesized by gram-negative bacteria with various biological activities such as antimicrobial, antiviral, and anticancer activities. VioD is a key oxygenase converting protodeoxyviolaceinic acid (PDVA) to protoviolaceinic acid in violacein biosynthesis. To elucidate the catalytic mechanism of VioD, here, we resolved two crystal structures of VioD, a binary complex structure containing VioD and a FAD and a ternary complex structure composed of VioD, a FAD and a 2-ethyl-1-hexanol (EHN). Structural analysis revealed a deep funnel like binding pocket with wide entrance, this pocket is positively charged. The EHN is located at the deep bottom of the binding pocket near isoalloxazine ring. Further docking simulation help us to propose the mechanism of the hydroxylation of the substrate catalyzed by VioD. Bioinformatic analysis suggested and emphasized the importance of the conserved residues involved in substrate binding. Our results provide a structural basis for the catalytic mechanism of VioD. This article is protected by copyright. All rights reserved.

Lipid environment determines the drug-stimulated ATPase activity of P-glycoprotein.

P-glycoprotein (Pgp), also known as ABCB1 or multidrug resistance protein 1 (MDR1), is expressed in the luminal membrane of the intestine, liver, and kidney where it regulates the absorption, distribution, metabolism, and excretion (ADME) of a wide range of xenobiotic compounds and metabolites, and at the blood-brain where it protects the sanctuary from uptake of toxic compounds and enhances excretion of β-amyloid and metabolites. Its expression in cancer cells has been linked to multidrug resistance against many chemotherapeutic agents leading to ineffective treatment and relapse. Besides chemotherapeutic drugs, lipids have been suggested to be transported by themselves or co-transported with drugs by Pgp. Here we tested the hypothesis that the plasma membrane lipid composition modulates how drugs interact with Pgp and may greatly affect both the affinity of the transport substrates and its maximum activity. Our general approach was to test varieties of synthetic lipid mixtures starting with the bilayer-forming phosphatidylcholine (PC) and supplementing with phospholipids carrying different head groups as well as cholesterol. We present the first results of Pgp interactions with PC, phosphatidylethanolamine (PE), sphingomyelin (SM), and cardiolipin which we found to act as positive effectors and increase the apparent affinity for substrates and increase Vmax, while others acted as negative effectors and decreased Vmax. The impact on the substrate binding and translocation mechanisms of Pgp will be discussed. The long-term goal is to establish a stable source of synthetic phospholipid mixtures supplemented with cholesterol that mimic Pgp activity in its native environment in the human body.

A Combined Computational–Experimental Study on the Substrate Binding and Reaction Mechanism of Salicylic Acid Decarboxylase

Salicylic acid decarboxylase (SDC) from the amidohydrolase superfamily (AHS) catalyzes the reversible decarboxylation of salicylic acid to form phenol. In this study, the substrate binding mode and reaction mechanism of SDC were investigated using computational and crystallographic methods. Quantum chemical calculations show that the enzyme follows the general mechanism of AHS decarboxylases. Namely, the reaction begins with proton transfer from a metal-coordinated aspartic acid residue (Asp298 in SDC) to the C1 of salicylic acid, which is followed by the C–C bond cleavage, to generate the phenol product and release CO2. Interestingly, the calculations show that SDC is a Mg-dependent enzyme rather than the previously proposed Zn-dependent, and the substrate is shown to be bidentately coordinated to the metal center in the catalysis, which is also different from the previous proposal. These predictions are corroborated by the crystal structure of SDC solved in complex with the substrate analogue 2-nitrophenol. The mechanistic insights into SDC in the present study provide important information for the rational design of the enzyme.

Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.

Enhancement of the performance of the GH75 family chitosanases by fusing a carbohydrate binding module and insights into their substrate binding mechanisms

Chitooligosaccharides (COSs) with relatively higher degrees of polymerization (DP) exhibited better biological properties than COSs with lower DP. Here, we demonstrated a potential strategy to enhance the affinity, activity, and thermostability of the chitosanase Csn75 from Aspergillus fumigatus CJ22-326 through fusion of a carbohydrate binding module (CBM) that can specifically bind to chitosan. Compared with the Csn75, the specific enzyme activity of the Csn75-CBM32 and the Csn75-2CBM32 had increased by 59.18% and 14.29%, respectively. The free energy (ΔG), enthalpy (ΔH), and entropy (-TΔS) of the three enzymes (Csn75, Csn75-CBM32, and Csn75-2CBM32) toward (GlcN)6 was (−7.19, −7.54, and −8.08 kcal/mol), (−5.37, 1.75, and 0.86 kcal/mol), and (1.45, 9.27, and 8.03 kcal/mol), respectively. The substrate binding of the Csn75 and the fusion enzymes (Csn75-CBM32 and Csn75-2CBM32) were mainly driven by the hydrophobic (entropy effect) and favorable intermolecular forces (enthalpy contribution), respectively. Moreover, the DP of COSs in the final hydrolysate of Csn75 was 2–4, but that of Csn75-CBM32 and Csn75-2CBM32 was 3–5. This strategy provides a potential alternative for the bioproduction of desirable COSs to meet the requirement of application in food systems.

The ArsI C-As lyase: Elucidating the catalytic mechanism of degradation of organoarsenicals

Organoarsenicals such as monosodium methylarsenate (MSMA or MAs(V)) and roxarsone (4–hydroxyl-3-nitrophenylarsenate or Rox(V)) have been extensively used as herbicides and growth enhancers for poultry, respectively. Degradation of organoarsenicals to inorganic arsenite (As(III)) contaminates crops and drinking water. One such process is catalyzed by the bacterial enzyme ArsI, whose gene is found in many soil bacteria. ArsI is a non-heme ferrous iron (Fe(II))-dependent dioxygenase that catalyzes oxygen-dependent cleavage of the carbon‑arsenic (C-As) bond in trivalent organoarsenicals, degrading them to inorganic As(III). From previous crystal structures of ArsI, we predicted that a loop-gating mechanism controls the catalytic reaction. Understanding the catalytic mechanism of ArsI requires knowledge of the mechanisms of substrate binding and activation of dioxygen. Here we report new ArsI structures with bound Rox(III) and mutant enzymes with alteration of active site residues. Our results elucidate steps in the catalytic cycle of this novel dioxygenase and enhance understanding of the recycling of environmental organoarsenicals.

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3–4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

Crystal structures of PigF, an O-methyltransferase involved in the prodigiosin synthetic pathway, reveal an induced-fit substrate-recognition mechanism.

Prodigiosin, a red linear tripyrrole pigment, is a typical secondary metabolite with numerous biological functions, such as anticancer, antibacterial and immunosuppressant activities, and is synthesized through a bifurcated biosynthesis pathway from 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amylpyrrole (MAP). The last step in the biosynthetic pathway of MBC is catalysed by PigF, which transfers a methyl group to 4-hydroxy-2,20-bipyrrole-5-carbaldehyde (HBC) to form the final product MBC. However, the catalytic mechanism of PigF is still elusive. In this study, crystal structures of apo PigF and S-adenosylhomocysteine (SAH)-bound PigF were determined. PigF forms a homodimer and each monomer consists of two domains: a C-terminal catalytic domain and an N-terminal dimerization domain. Apo PigF adopts an open conformation, while the structure of the complex with the product SAH adopts a closed conformation. The binding of SAH induces dramatic conformational changes of PigF, suggesting an induced-fit substrate-binding mechanism. Further structural comparison suggests that this induced-fit substrate-recognition mechanism may generally exist in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) that are crucial for enzyme activity. The essential function of His247 and Asp248 and structure analysis suggests that both residues are involved in activation of the HBC substrate of PigF. The invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. This study provides new insight into the catalytic mechanism of PigF, reveals an induced-fit substrate-recognition model for PigF and broadens the understanding of O-methyltransferases.

Membrane recognition and substrate binding mechanism of PIP4Kγ kinase from molecular simulations

PIP4Kγ belongs to a family of lipid kinases that use phosphatidylinositol-5-phosphate (PI5P) as a substrate to produce phosphatidylinositol-4,5-biphosphate, and has recently emerged as a neurodegenerative disease therapeutic target. However, the underlying structural basis of membrane recognition and substrate binding largely remains unknown as there is no crystal structure of the substrate-bound lipid kinase. Moreover, the key structural elements suggested to be involved in substrate binding and recognition, the DLKGS motif and the activation loop region as well as the ATP binding site, are not resolved in the PIP4Kγ crystal structure.

Molecular mechanism for conformational activation of SARS-CoV-2 RNA polymerase by nucleoside triphosphate

It has been shown that the nucleotide analogs such as remdesivir and molnupiravir are promising drug candidates to treat COVID-19 infection, by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Characterizing the molecular mechanism of RNA replication by RdRp is crucial to understand how these inhibitors work. Here we carried out molecular dynamics simulations coupled with enhanced sampling techniques to study the substrate binding mechanism and conformational dynamics of the SARS-CoV-2 RdRp complex based on recently available cryo-EM structures. Our results reveal the transition pathways for active site closure, which is required for substrate catalysis. Structural bioinformatics analysis indicates such motions exist in other homologous viral polymerases. Furthermore, we demonstrate that the RNA-binding cleft motion is coupled to the coordination of two magnesium ions at the active site. Motif G is found to play a key role in nucleotide positioning during the activation process. By applying the Gaussian accelerated molecular dynamics (GaMD) to enhance the sampling of the substrate dynamics, we find a key intermediate state that precedes the tight binding of the nucleoside triphosphate. The simulations provide metastable RdRp conformations at the active site, which can help the drug discovery using various approved nucleotide inhibitors. It has been shown that the nucleotide analogs such as remdesivir and molnupiravir are promising drug candidates to treat COVID-19 infection, by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Characterizing the molecular mechanism of RNA replication by RdRp is crucial to understand how these inhibitors work. Here we carried out molecular dynamics simulations coupled with enhanced sampling techniques to study the substrate binding mechanism and conformational dynamics of the SARS-CoV-2 RdRp complex based on recently available cryo-EM structures. Our results reveal the transition pathways for active site closure, which is required for substrate catalysis. Structural bioinformatics analysis indicates such motions exist in other homologous viral polymerases. Furthermore, we demonstrate that the RNA-binding cleft motion is coupled to the coordination of two magnesium ions at the active site. Motif G is found to play a key role in nucleotide positioning during the activation process. By applying the Gaussian accelerated molecular dynamics (GaMD) to enhance the sampling of the substrate dynamics, we find a key intermediate state that precedes the tight binding of the nucleoside triphosphate. The simulations provide metastable RdRp conformations at the active site, which can help the drug discovery using various approved nucleotide inhibitors.

Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2.

Neutral amino acid transporters ASCT1 and ASCT2 are two SLC1 (solute carrier 1) family subtypes, which are specific for neutral amino acids. The other members of the SLC1 family are acidic amino acid transporters (EAATs 1–5). While the functional similarities and differences between the EAATs have been well studied, less is known about how the subtypes ASCT1 and 2 differ in kinetics and function. Here, by performing comprehensive electrophysiological analysis, we identified similarities and differences between these subtypes, as well as novel functional properties, such as apparent substrate affinities of the inward-facing conformation (in the range of 70 μM for L-serine as the substrate). Key findings were: ASCT1 has a higher apparent affinity for Na+, as well as a larger [Na+] dependence of substrate affinity compared to ASCT2. However, the general sequential Na+/substrate binding mechanism with at least one Na+ binding first, followed by amino acid substrate, followed by at least one more Na+ ion, appears to be conserved between the two subtypes. In addition, the first Na+ binding step, presumably to the Na3 site, occurs with high apparent affinity (<1 mM) in both transporters. In addition, ASCT1 and 2 show different substrate selectivities, where ASCT1 does not respond to extracellular glutamine. Finally, in both transporters, we measured rapid, capacitive charge movements upon application and removal of amino acid, due to rearrangement of the translocation equilibrium. This charge movement decays rapidly, with a time constant of 4–5 ms and recovers with a time constant in the 15 ms range after substrate removal. This places a lower limit on the turnover rate of amino acid exchange by these two transporters of 60–80 s−1.