Orders Of Magnitude Higher Affinity Research Articles

Determination of Calcium in Meat Products by Automatic Titration with 1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic Acid.

Mechanically separated meat (MSM) is a by-product of the poultry industry that requires routine quality assessment. Calcium content is an indirect indicator of bone debris in MSM but is difficult to determine by EDTA titration due to the poor solubility of calcium phosphate. Therefore, 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid was used instead, which has two orders of magnitude higher affinity for calcium ions. In addition, the auxiliary complexing agents triethanolamine and Arsenazo III, an indicator that is sensitive to low calcium concentrations, were used. Automatic titration endpoint detection was performed using an immersion probe at 660 nm. It has been shown that the color change in Arsenazo III can also be read with an RGB camera. The CDTA titration procedure has been tested on commercial Bologna-type sausages and the results were in line with AAS and ICP reference data. The content of calcium in sausages turned out to be very diverse and weakly correlated with the content of MSM. The tested MSM samples had a wide range of calcium content: from 62 to 2833 ppm. Calcium-rich poultry by-products include fat and skin (115 to 412 ppm), articular cartilage (1069 to 1704 ppm), and tendons (532 to 34,539 ppm). The CDTA titration procedure is fully suitable for small meat processing plants due to its simplicity of use and low cost.

Galectin-9 as a Potential Modulator of Lymphocyte Adhesion to Endothelium via Binding to Blood Group H Glycan.

The recruitment of leukocytes from blood is one of the most important cellular processes in response to tissue damage and inflammation. This multi-step process includes rolling leukocytes and their adhesion to endothelial cells (EC), culminating in crossing the EC barrier to reach the inflamed tissue. Galectin-8 and galectin-9 expressed on the immune system cells are part of this process and can induce cell adhesion via binding to oligolactosamine glycans. Similarly, these galectins have an order of magnitude higher affinity towards glycans of the ABH blood group system, widely represented on ECs. However, the roles of gal-8 and gal-9 as mediators of adhesion to endothelial ABH antigens are practically unknown. In this work, we investigated whether H antigen-gal-9-mediated adhesion occurred between Jurkat cells (of lymphocytic origin and known to have gal-9) and EA.hy 926 cells (immortalized endothelial cells and known to have blood group H antigen). Baseline experiments showed that Jurkat cells adhered to EA.hy 926 cells; however when these EA.hy 926 cells were defucosylated (despite the unmasking of lactosamine chains), adherence was abolished. Restoration of fucosylation by insertion of synthetic glycolipids in the form of H (type 2) trisaccharide Fucα1-2Galβ1-4GlcNAc restored adhesion. The degree of lymphocyte adhesion to native and the "H-restored" (glycolipid-loaded) EA.hy 926 cells was comparable. If this gal-9/H (type 2) interaction is similar to processes that occur in vivo, this suggests that only the short (trisaccharide) H glycan on ECs is required.

Mutant Flavin-Based Fluorescent Protein Sensors for Detecting Intracellular Zinc and Copper in Escherichia coli.

Flavin-based fluorescent proteins (FbFPs) are a class of fluorescent reporters that undergo oxygen-independent fluorophore incorporation, which is an important advantage over green fluorescent proteins (GFPs) and mFruits. A FbFP derived from Chlamydomonas reinhardtii (CreiLOV) is a promising platform for designing new metal sensors. Some FbFPs are intrinsically quenched by metal ions, but the question of where metals bind and how to tune metal affinity has not been addressed. We used site-directed mutagenesis of CreiLOV to probe a hypothesized copper(II) binding site that led to fluorescence quenching. Most mutations changed the fluorescence quenching level, supporting the proposed site. One key mutation introducing a second cysteine residue in place of asparagine (CreiLOVN41C) significantly altered metal affinity and selectivity, yielding a zinc sensor. The fluorescence intensity and lifetime of CreiLOVN41C were reversibly quenched by Zn2+ ions with a biologically relevant affinity (apparent dissociation constant, Kd, of 1 nM). Copper quenching of CreiLOVN41C was retained but with several orders of magnitude higher affinity than CreiLOV (Kd = 0.066 fM for Cu2+, 5.4 fM for Cu+) and partial reversibility. We also show that CreiLOVN41C is an excellent intensity- and lifetime-based zinc sensor in aerobic and anaerobic live bacterial cells. Zn2+-induced fluorescence quenching is reversible over several cycles in Escherichia coli cell suspensions and can be imaged by fluorescence microscopy. CreiLOVN41C is a novel oxygen-independent metal sensor that significantly expands the current fluorescent protein-based toolbox of metal sensors and will allow for studies of anaerobic and low oxygen systems previously precluded by the use of oxygen-dependent GFPs.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

DCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

SUMMARYBifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη.

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

The first MultiTEP‐based conjugated vaccine targeting pyroglutamated pE3‐Aβ is immunogenic and effective in a 5xFAD animal model

AbstractBackgroundMany ongoing studies for AD immunotherapy are focused on the immunodominant N‐terminus of Aβ. This region is often truncated and post‐translationally modified in amyloid. The pyroglutamate modified variant of Aβ40/42 (pE3‐Aβ) is more neurotoxic and aggregation‐prone and is thought to play an important role in disease onset. We have previously demonstrated that our proprietary vaccine platform MultiTEP is extremely immunogenic in various mammals, including disease models of AD and aged monkeys, and can be used to generate a robust immune response towards various targets. Based on these we developed a MutiTEP‐based vaccine targeting pE3‐Aβ.MethodA genetically engineered variant of MultiTEP was generated to enable chemoselective bioconjugation of synthetic peptides under mild aqueous conditions. The peptide‐protein stoichiometry was selected at 3:1 based on previous data. C57Bl6 and 5xFAD mice were monthly immunized with pE3‐Aβ‐MultiTEP adjuvanted vaccine, AV‐1986R. Antibody titers were tested via ELISA on plates coated with pE3‐Aβ42 and unmodified Aβ42 as control. The reduction of brain pathology in 5xFAD was assessed via biochemical assessment (MSD ELISA) as well as IHC and confocal microscopy using various commercial antibodies. The binding of immune sera to brain sections from non‐AD, MCI, and AD cases was examined.ResultThe AV‐1986R generates a robust immune response in transgenic and WT mice after two immunizations. The generated antibodies exhibit three orders of magnitude higher affinity (one to a million avg endpoint titer) to the pE3‐Aβ compared to Aβ42 and recognized Aβ plaques in brain tissue slices from both transgenic animals and AD cases. Importantly, vaccination significantly reduced AD‐like pathology in 5xFAD mice.ConclusionAV‐1986R is immunogenic and effective. A recent report from Elly‐Lilly about the pE3‐Aβ specific monoclonal antibody donanemab shows that treatment slows down the cognitive decline in humans. While this is promising and re‐affirms the selection of pE3‐Aβ as a therapeutic target, the regular injections of monoclonal antibodies are extremely expensive and have low patient compliance due to the frequent and invasive nature of the treatment. We suggest using active vaccine as a feasible alternative, allowing the maintenance of a constant level of therapeutic antibodies with minimal costs and infrequent vaccine injections.

Importance of protein intrinsic conformational dynamics and transient nature of non-covalent interactions in ligand binding affinity

We have recently identified BEN1 as a protein interactor of seryl-tRNA synthetase (SerRS) from model plant Arabidopsis thaliana. BEN1 contains an NADP+ binding domain and possesses acidic N-terminal extension essential for interaction with A. thaliana SerRS. This extension, specific for BEN1 homologues from Brassicaceae family, is solvent-exposed and distant to the nucleotide-binding site. We prepared a truncated BEN1 variant ΔN17BEN1 lacking the first 17 amino acid of this N-terminal extension as well as full-length BEN1 to investigate how the truncation affects the binding affinity towards coenzyme NADP+. By performing microscale thermophoresis (MST) experiments we have shown that both BEN1 variants bind the NADP+ cofactor, however, truncated BEN1 showed 34-fold higher affinity towards NADP+ indicating that its core protein structure is not just preserved but it binds NADP+ even stronger. To further corroborate the obtained results, we opted for a computational approach based on classical molecular dynamics simulations of both complexes. Our results have shown that both truncated and intact BEN1 variants form the same number of interactions with the NADP+ cofactor; however, it was the interaction occupancy that was affected. Namely, three independent MD simulations showed that the ΔN17BEN1 variant in complex with NADP+ has significantly higher interaction occupancy thus binds NADP+ with more than one order of magnitude higher affinity. Contrary to our expectations, the truncation of this distant region that does not communicate with the nucleotide-binding site didn't result in the gain of interaction but affected the intrinsic conformational dynamics which in turn fine-tuned the binding affinity by increasing the interaction occupancy and strength of the key conserved cation-π interaction between Arg69 and adenine of NADP+ and hydrogen bond between Ser244 and phosphate of NADP+.

Abstract LB-382: Engineered NK92MICD64 cell docking platform as a combination therapy approach for prostate cancer

Abstract Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill virally infected or malignant cells. Targeting NK cells to solid tumors can be improved by engineering NK cells to express CD64 (FcγR1), a high affinity receptor for human IgG Fc, in combination with therapeutic monoclonal antibodies (mAbs). CD64 can capture soluble mAbs with two to three orders of magnitude higher affinity than CD16A (FcγIIIA) and mediates tumor cell killing when anti-tumor mAb is bound. This docking platform allows for switchable targeting elements to effectively direct the NK cells to multiple tumor antigens. We are targeting several relevant prostate cancer (PCa) antigens including TROP2, a transmembrane glycoprotein that is overexpressed in metastatic PCa, and FAP, a membrane bound serine protease expressed by cancer-associated fibroblasts in the tumor microenvironment. For all cytotoxicity experiments, we used an immortalized human PCa stromal cell line that highly expresses FAP (hPrCSC-44) and a human PCa cell line that highly expresses TROP2 (DU145). Cell killing was measured using the Delfia EuTDA cytotoxicity assay. First, we tested for mediation of NK92MICD64 cell antibody dependent cell cytotoxicity (ADCC) using a titration of anti-FAP or anti-TROP2 mAb (5 to 0 µg/mL) and saw a concentration dependent effect on cell killing. Next, CD64 capture of soluble anti-FAP or anti-TROP2 mAb was tested by flow cytometry. Over 98% of the NK92MICD64 cells incubated with the therapeutic mAbs were positively stained, indicating that the mAbs dock to CD64 and could direct tumor cell killing. Therapeutic efficacy of anti-FAP or anti-TROP2 mAb bound to NK92MICD64 cells (αFAP-NK92MICD64 and αTROP2-NK92MICD64) was tested by incubating the effector cells with hPrCSC-44 or DU145 cells at several effector:target (E:T) ratios. Potent cytotoxicity was observed and the % specific release was significantly higher than the NK92MICD64 cells alone. Cytotoxicity dropped to baseline levels when the anti-tumor mAb was switched to an isotype control, indicating that the mAb-directed cell killing occurs by an antigen-selective mechanism. After seeing the success of the monotherapies, we wanted to evaluate αFAP-NK92MICD64 and αTROP2-NK92MICD64 cells as a combination therapy. Previous studies have reported a synergistic effect of combination therapies that target both the tumor stroma and malignant cells. To test our approach, hPrCSC-44 and DU145 cells were co-incubated with αFAP-NK92MICD64 and αTROP2-NK92MICD64 cells at several E:T ratios. We observed potent cytotoxicity and % specific release was significantly higher than the NK92MICD64 cells alone. In conclusion, CD64 on NK92MI cells facilitated ADCC and cytokine production demonstrating functional activity in our prostate cancer models. The use of CD64 as a docking platform for therapeutic mAbs was shown to effectively mediate tumor cell killing. Together, these data suggest the potential for our combination cell therapy to be developed as a new therapeutic approach for PCa. Citation Format: Hallie M. Hintz, Aaron M. LeBeau. Engineered NK92MICD64 cell docking platform as a combination therapy approach for prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-382.

ATP-binding affinity of the ε subunit of thermophilic F1-ATPase under label-free conditions

The ε subunits of several bacterial F1-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F1-ATPase from thermophilic Bacillus sp. PS3 (TF1). We previously reported that the dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C is 4.3 μM by measuring changes in the fluorescence of the dye attached to the ε subunit (Kato, S. et al. (2007) J. Biol. Chem.282, 37618). However, we have recently noticed that this varies with the dye used. In this report, to determine the affinity for ATP under label-free conditions, we have measured the competitive displacement of 2′(3′)-O-N′-methylaniloyl-aminoadenosine-5′-triphosphate (Mant-ATP), a fluorescent analog of ATP, by ATP. The dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C was determined to be 0.29 μM, which is one order of magnitude higher affinity than previously reported values.

Interrogating Intracellular Zinc Chemistry with a Long Stokes Shift Zinc Probe ZincBY-4.

Previous work has shown that fluctuations in zinc content and subcellular localization play key roles in regulating cell cycle progression; however, a deep mechanistic understanding requires the determination of when, where, and how labile zinc pools are concentrated into or released from stores. Labile zinc ions can be difficult to detect with probes that require hydrolysis of toxic protecting groups or application at high concentrations that negatively impact cell function. We previously reported a BODIPY-based zinc probe, ZincBY-1, that can be used at working concentrations that are 20-200-fold lower than concentrations employed with other probes. To better understand how zinc pools can be visualized at such low probe concentrations, we modulated the photophysical properties via changes at the 5-position of the BODIPY core. One of these, ZincBY-4, exhibits an order of magnitude higher affinity for zinc, an 8-fold increase in brightness in response to zinc, and a 100 nm Stokes shift within cells. The larger Stokes shift of ZincBY-4 presents a unique opportunity for simultaneous imaging with GFP or fluorescein sensors upon single excitation. Finally, by creating a proxy for the cellular environment in spectrometer experiments, we show that the ZincBY series are highly effective at 50 nM because they can pass membranes and accumulate in regions of high zinc concentration within a cell. These features of the ZincBY probe class have widespread applications in imaging and for understanding the regulatory roles of zinc fluxes in live cells.

Toxins for decoding interface selectivity in nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play crucial roles in neurotransmission and regulate complex processes in brain functions, including anxiety, learning and memory, food intake, drug addiction, cognition and nociception. To perform these and other functions, a diverse array of nAChR subtypes are generated by homomeric or heteromeric assembly of 17 homologous nAChR subunits. Agonists, acetylcholine and nicotine, bind to the interface formed between two α subunits and between α and non-α subunits to activate the nAChR and allow cation influx. The diversity of subunit interfaces determines the channel properties, the responses to different agonists/antagonists, desensitization and downstream signaling and thus, define specialized properties and functions. Over the last several decades, snake venom neurotoxins have contributed to the purification, localization and characterization of molecular details of various nAChRs. Utkin et al. have described the purification and characterization of αδ-bungarotoxins, a novel class of neurotoxins in a recent paper published in the Biochemical Journal [Biochem. J. (2019) 476, 1285-1302]. These toxins from Bungarus candidus venom preferably bind to α-δ site with two orders of magnitude higher affinity compared with α-γ or α-ε sites. The subtle changes in the structure of αδ-bungarotoxins led to variation in interface selectivity. Such new classes of antagonists will offer us great opportunity to delineate the pharmacophores and design new highly selective antagonists. Thus, their findings provide new impetus to re-evaluate molecular details of pharmacological properties of α-neurotoxins with careful consideration towards subtype-, interface- and species-selectivity.

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.

The 1-β-methyl group confers a lower affinity of l,d-transpeptidase LdtMt2 for ertapenem than for imipenem

L,D-transpeptidases, widely distributed in bacteria and even in the difficult-to-treat ESKAPE pathogens, can confer antibacterial resistance against the traditional β-lactam antibiotics through bypass of the 4 → 3 transpeptide linkage. LdtMt2, a l,d-transpeptidase in Mycobacteria tuberculosis, is essential for bacterial virulence and is considered as a potential anti-tuberculosis target inhibited by carbapenems. Diverse interaction modes between carbapenems and LdtMt2 have been reported, there are only limited evidences to validate those interaction modes. Herein, we identified the stable binding states of two carbapenems, imipenem and ertapenem, via crystallographic and biochemical studies, discovered that they adopt similar binding conformations. We further demonstrate the absence of the 1-β-methyl group in imipenem and the presence of both Y308 and Y318 residues in LdtMt2 synergistically resulted in one order of magnitude higher affinity for imipenem than ertapenem. Our study provides a structural basis for the rational drug design and evolvement of novel carbapenems against bacterial L,D-transpeptidases.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display.

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.

Three-dimensional context rather than NLS amino acid sequence determines importin \u03b1 subtype specificity for RCC1

Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3. This pathway is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Here we identify the mechanisms of importin α3 selectivity for RCC1. We find this isoform binds RCC1 with one order of magnitude higher affinity than the generic importin α1, although the two isoforms share an identical NLS-binding groove. Importin α3 uses its greater conformational flexibility to wedge the RCC1 β-propeller flanking the NLS against its lateral surface, preventing steric clashes with its Armadillo-core. Removing the β-propeller, or inserting a linker between NLS and β-propeller, disrupts specificity for importin α3, demonstrating the structural context rather than NLS sequence determines selectivity for isoform 3. We propose importin α3 evolved to recognize topologically complex NLSs that lie next to bulky domains or are masked by quaternary structures.

Incorporating an allosteric regulatory site in an antibody through backbone design.

Allosteric regulation underlies living cells' ability to sense changes in nutrient and signaling-molecule concentrations, but the ability to computationally design allosteric regulation into non-allosteric proteins has been elusive. Allosteric-site design is complicated by the requirement to encode the relative stabilities of active and inactive conformations of the same protein in the presence and absence of both ligand and effector. To address this challenge, we used Rosetta to design the backbone of the flexible heavy-chain complementarity-determining region 3 (HCDR3), and used geometric matching and sequence optimization to place a Zn2+ -coordination site in a fluorescein-binding antibody. We predicted that due to HCDR3's flexibility, the fluorescein-binding pocket would configure properly only upon Zn2+ application. We found that regulation by Zn2+ was reversible and sensitive to the divalent ion's identity, and came at the cost of reduced antibody stability and fluorescein-binding affinity. Fluorescein bound at an order of magnitude higher affinity in the presence of Zn2+ than in its absence, and the increase in fluorescein affinity was due almost entirely to faster fluorescein on-rate, suggesting that Zn2+ preorganized the antibody for fluorescein binding. Mutation analysis demonstrated the extreme sensitivity of Zn2+ regulation on the atomic details in and around the metal-coordination site. The designed antibody could serve to study how allosteric regulation evolved from non-allosteric binding proteins, and suggests a way to designing molecular sensors for environmental and biomedical targets.

An Open and Shut Case: The Interaction of Magnesium with MST Enzymes.

The shikimate pathway of bacteria, fungi, and plants generates chorismate, which is drawn into biosynthetic pathways that form aromatic amino acids and other important metabolites, including folates, menaquinone, and siderophores. Many of the pathways initiated at this branch point transform chorismate using an MST enzyme. The MST enzymes (menaquinone, siderophore, and tryptophan biosynthetic enzymes) are structurally homologous and magnesium-dependent, and all perform similar chemical permutations to chorismate by nucleophilic addition (hydroxyl or amine) at the 2-position of the ring, inducing displacement of the 4-hydroxyl. The isomerase enzymes release isochorismate or aminodeoxychorismate as the product, while the synthase enzymes also have lyase activity that displaces pyruvate to form either salicylate or anthranilate. This has led to the hypothesis that the isomerase and lyase activities performed by the MST enzymes are functionally conserved. Here we have developed tailored pre-steady-state approaches to establish the kinetic mechanisms of the isochorismate and salicylate synthase enzymes of siderophore biosynthesis. Our data are centered on the role of magnesium ions, which inhibit the isochorismate synthase enzymes but not the salicylate synthase enzymes. Prior structural data have suggested that binding of the metal ion occludes access or egress of substrates. Our kinetic data indicate that for the production of isochorismate, a high magnesium ion concentration suppresses the rate of release of product, accounting for the observed inhibition and establishing the basis of the ordered-addition kinetic mechanism. Moreover, we show that isochorismate is channeled through the synthase reaction as an intermediate that is retained in the active site by the magnesium ion. Indeed, the lyase-active enzyme has 3 orders of magnitude higher affinity for the isochorismate complex relative to the chorismate complex. Apparent negative-feedback inhibition by ferrous ions is documented at nanomolar concentrations, which is a potentially physiologically relevant mode of regulation for siderophore biosynthesis in vivo.

High affinity nucleotide-binding mutant of the ε subunit of thermophilic F1-ATPase

Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16 and 53 μM, respectively. Thus, the mutant would be a good platform for various types of nucleotide biosensor with appropriate modifications.

Species-Specific Structural and Functional Divergence of α-Crystallins: Zebrafish αBa- and Rodent αA(ins)-Crystallin Encode Activated Chaperones.

In addition to contributing to lens optical properties, the α-crystallins are small heat shock proteins that possess chaperone activity and are predicted to bind and sequester destabilized proteins to delay cataract formation. The current model of α-crystallin chaperone mechanism envisions a transition from the native oligomer to an activated form that has higher affinity to non-native states of the substrate. Previous studies have suggested that this oligomeric plasticity is encoded in the primary sequence and controls access to high affinity binding sites within the N-terminal domain. Here, we further examined the role of sequence variation in the context of species-specific α-crystallins from rat and zebrafish. Alternative splicing of the αA gene in rodents produces αA(ins), which is distinguished by a longer N-terminal domain. The zebrafish genome includes duplicate αB-crystallin genes, αBa and αBb, which display divergent primary sequence and tissue expression patterns. Equilibrium binding experiments were employed to quantitatively define chaperone interactions with a destabilized model substrate, T4 lysozyme. In combination with multiangle light scattering, we show that rat αA(ins) and zebrafish α-crystallins display distinct global structural properties and chaperone activities. Notably, we find that αA(ins) and αBa demonstrate substantially enhanced chaperone function relative to other α-crystallins, binding the same substrate more than 2 orders of magnitude higher affinity and mimicking the activity of fully activated mammalian small heat shock proteins. These results emphasize the role of sequence divergence as an evolutionary strategy to tune chaperone function to the requirements of the tissues and organisms in which they are expressed.