Β2-β3 Loop Research Articles

Kunitz-type trypsin inhibitor from durian (Durio zibethinus) employs a distinct loop for trypsin inhibition.

Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors.

Structural basis of nucleic acid recognition by the N-terminal cold shock domain of the plant glycine-rich protein AtGRP2

AtGRP2 is a glycine-rich, RNA-binding protein that plays pivotal roles in abiotic stress response and flowering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal CCHC-type zinc knuckles interspersed with glycine-rich regions. Here, we investigated the structure, dynamics, and nucleic acid binding properties of AtGRP2-CSD. The 2D [1H,15N] HSQC spectrum of AtGRP2-CSD1-79 revealed the presence of a partially folded intermediate in equilibrium with the folded state. The addition of eleven residues at the C-terminus stabilized the folded conformation. The three-dimensional structure of AtGRP2-CSD1-90 unveiled a β-barrel composed of five antiparallel β-strands and a 310 helical turn, along with an ordered C-terminal extension, a conserved feature in eukaryotic CSDs. Direct contacts between the C-terminal extension and the β3-β4 loop further stabilized the CSD fold. AtGRP2-CSD1-90 exhibited nucleic acid binding via solvent-exposed residues on strands β2 and β3, as well as the β3-β4 loop, with higher affinity for DNA over RNA, particularly favoring pyrimidine-rich sequences. Furthermore, DNA binding induced rigidity in the β3-β4 loop, evidenced by 15N-{1H} NOE values. Mutation of residues W17, F26, and F37, in the central β-sheet, completely abolished DNA binding, highlighting the significance of π-stacking interactions in the binding mechanism. These results shed light on the mechanism of nucleic acid recognition employed by AtGRP2, creating a framework for the development of biotechnological strategies aimed at enhancing plant resistance to abiotic stresses.

Long Dynamic β1-β2 Loops in M. tb MazF Toxins Affect the Interaction Modes and Strengths of the Toxin-Antitoxin Pairs.

Tuberculosis is a worldwide plague caused by the pathogen Mycobacterium tuberculosis (M. tb). Toxin-antitoxin (TA) systems are genetic elements abundantly present in prokaryotic organisms and regulate important cellular processes. MazEF is a TA system implicated in the formation of "persisters cells" of M. tb, which contain more than 10 such members. However, the exact function and inhibition mode of each MazF are not fully understood. Here we report crystal structures of MazF-mt3 in its apo form and in complex with the C-terminal half of MazE-mt3. Structural analysis suggested that two long but disordered β1-β2 loops would interfere with the binding of the cognate MazE-mt3 antitoxin. Similar loops are also present in the MazF-mt1 and -mt9 but are sustainably shortened in other M. tb MazF members, and these TA pairs behave distinctly in terms of their binding modes and their RNase activities. Systematic crystallographic and biochemical studies further revealed that the biochemical activities of M. tb toxins were combined results between the interferences from the characteristic loops and the electrostatic interactions between the cognate TA pairs. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F TA pairs, which facilitate the structure-based peptide designs.

Understanding and manipulating the recognition of necrosis-inducing secreted protein 1 (NIS1) by BRI1-associated receptor kinase 1 (BAK1)

Necrosis-inducing secreted protein 1 (NIS1) is a core effector of Ascomycota and Basidiomycota fungi. They inhibit the immune responses of host plants mainly through interaction with the multi-functional coreceptor BRI1-associated receptor kinase 1 (BAK1). However, the structural mechanism of the NIS1 family and how they are recognized by BAK1 are unknown. Herein, we report the first crystal structure of the NIS1 family protein, the Magnaporthe oryzae NIS1 (MoNIS1), analyze the recognition mechanism of NIS1s by BAK1, and explore regulation of the NIS1-BAK1 interaction by a chemical compound. MoNIS1 exists as a β barrel formed by eight β strands, a folding mode that has not been reported. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) assay suggested that β4-β5 loop and β5 strand of MoNIS1 participate in OsBAK1 interaction, which was supported by further single-point mutational assays. For OsBAK1, HDX-MS assay suggested four regions involved in MoNIS1 interaction. Additionally, we identified a compound that blocks MoNIS1-OsBAK1 interaction in vitro and inhibits the virulence of M. oryzae on rice. Collectively, we determined the first structure of NIS1 family effectors, presented the recognition mechanism of NIS1 by BAK1, and showed that blocking NIS1-BAK1 interaction could be a new target for fungicide development.

Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k2/KS, by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k2, and the weakening of the binding affinity, i.e., an increase in the substrate constant, KS. A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2A501V variant exhibits a higher k2/KS value, whereas for PBP2A501R and PBP2A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k2/KS changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the β-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k2/KS value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the β3-β4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k2/KS value. Thus, the experimentally observed decrease in the k2/KS in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k2, and the increase in KS.

Characterization of the extended substrate spectrum of the class A β-lactamase CESS-1 from Stenotrophomonas sp. and structure-based investigation into its substrate preference

Stenotrophomonas spp. intrinsically resistant to many β-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A β-lactamase. The goal of this study was to reveal biochemical and structural characteristics of CESS-1. The hydrolytic activities of CESS-1 towards penicillins (penicillin G and ampicillin), cephalosporins (cephalexin, cefaclor, and cefotaxime), and carbapenems (imipenem and meropenem) was spectrophotometrically monitored. Structural information on E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin were determined by X-ray crystallography. CESS-1 displayed hydrolytic activities toward penicillins and cephalosporins, with negligible activity toward carbapenems. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward them were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were 1249.6 s-1, 204.3 s-1, and 69.8 s-1, respectively, with the accompanying KM values of 287.6 μM, 236.7 μM, and 28.8 μM, respectively. CESS-1 was able to discriminate between cefaclor and cephalexin with a single structural difference at C3 position: -Cl (cefaclor) and -CH3 (cephalexin). Structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin, revealed that cooperative positional changes in the R1 side chain of substrates and their interaction with the β5-β6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar β-lactam antibiotics.

Role of amino acid 159 in carbapenem and temocillin hydrolysis of OXA-933, a novel OXA-48 variant.

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The blaOXA-933 gene was borne on Tn2208, a 2,696-bp composite transposon made of two IS1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the blaOXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the blaOXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it.

Molecular Dynamics Simulations of Claudin-10a and -10b Ion Channels: With Similar Architecture, Different Pore Linings Determine the Opposite Charge Selectivity.

Claudin polymers constitute the tight junction (TJ) backbone that forms paracellular barriers, at least for bigger solutes. While some claudins also seal the barrier for small electrolytes, others form ion channels. For cation-selective claudin-15 and claudin-10b, structural models of channels embedded in homo-polymeric strands have been suggested. Here, we generated a model for the prototypic anion-selective claudin-10a channel. Based on previously established claudin-10b models, dodecamer homology models of claudin-10a embedded in two membranes were analyzed by molecular dynamics simulations. The results indicate that both claudin-10 isoforms share the same strand and channel architecture: Sidewise unsealed tetrameric pore scaffolds are interlocked with adjacent pores via the β1β2 loop of extracellular segment 1. This leads to TJ-like strands with claudin subunits arranged in four joined rows in two opposing membranes. Several but not all cis- and trans-interaction modes are indicated to be conserved among claudin-10a, -10b, and -15. However, pore-lining residues that differ between claudin-10a and -10b (i.e., R33/I35, A34/D36, K69/A71, N54/D56, H60/N62, R62/K64) result in opposite charge selectivity of channels. This was supported by electric field simulations for both claudins and is consistent with previous electrophysiological studies. In summary, for the first time, a structural and mechanistic model of complete and prototypic paracellular anion channels is provided. This improves understanding of epithelial paracellular transport.

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9’ hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.

Modulation of the conformational landscape of the PDZ3 domain by perturbation on a distal non-canonical α3 helix: decoding the microscopic mechanism of allostery in the PDZ3 domain.

While allosteric signal transduction is crucial for protein signaling and regulation, the dynamic process of allosteric communication remains poorly understood. The third PDZ domain (PDZ stands for the common structural domain shared by the postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (ZO-1)) serves as a classic example of a single-domain allosteric protein, demonstrating a long-range coupling between the C-terminal α helix (known as the α3 helix) and ligand binding. A molecular level understanding of how the α3 helix modulates the ligand binding affinity of the PDZ3 domain is still lacking. In this study, extensive molecular dynamics simulations corroborated with principal component analysis (PCA), ligand binding free energy calculations, energetic frustration analysis and Markov state model analysis are employed to uncover such molecular details. We demonstrate the definite presence of a binding competent closed-like state in the conformational landscape of wild-type PDZ3. The population modulations of this closed state and other binding incompetent states in the landscape due to α3-truncation/mutation of PDZ3 are explored. A correlation between the closed state population and calculated binding free energy is established, which supports the conformation selection mechanism. Covariance analysis identified the presence of correlated motion between two distant loops (β1-β2 and β2-β3) in the wild-type PDZ3 system, which weakened due to truncation/mutation in the distant α3 helix. It has also been observed that whenever the α3 helix was perturbed, the β2-β3 loop got further away from the binding groove and it is found to be correlated with the binding free energy values. Energetic frustration analysis of the PDZ3 domain also showed that the β2-β3 loop is highly frustrated. Finally, MSM analysis revealed a relevant timescale (closed to open state transition), which is similar to the observed experimental signal transduction timescale for the system. These observations led to the conclusion that the distantly located α3 helix plays a pivotal role in regulating the conformational landscape of the PDZ3 domain, determining the ligand binding affinity and resulting in allosteric behavior of the domain.

Domain III β4-β5 Loop and β14-β15 Loop of Bacillus thuringiensis Vip3Aa Are Involved in Receptor Binding and Toxicity.

Vip3Aa, secreted by Bacillus thuringiensis, is effective at controlling major agricultural pests such as Spodoptera frugiperda. However, to control Vip3Aa resistance evolved in the field by different lepidoptera species, an in-depth study of sequence--structure--activity relationships is necessary to design new Vip3Aa variants. In this study, the four specific loops (β4-β5 loop, β9-β10 loop, β12-β13 loop, and β14-β15 loop) in domain III were selected and four loop mutants were constructed by replacing all residues in each specific loop with alanine. We obtained soluble proteins for three of the loop mutants, excluding the β9-β10 loop. These loop mutants have been characterized by toxicity bioassays against S. frugiperda, proteolytic processing, and receptor binding. These results indicate that the β4-β5 loop and β14-β15 loop are involved in receptor binding and Vip3Aa toxicity. Based on this, we constructed numerous mutants and obtained three single mutants (Vip3Aa-S366T, Vip3Aa-S366L, and Vip3Aa-R501A) that exhibited significantly increased toxicity of 2.61-fold, 3.39-fold, and 2.51-fold, respectively. Compared to Vip3Aa, the receptor affinity of Vip3Aa-S366T and Vip3Aa-S366L was significantly enhanced. Furthermore, we also analyzed and aligned the three-dimensional structures of the mutants and Vip3Aa. In summary, these results indicate that the loops in domain III have the potential to be targeted to enhance the insecticidal toxicity of the Vip3Aa protein.

Propagation of conformational instability in FK506-binding protein FKBP12

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long β4-β5 loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The β4-β5 loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the β4-β5 loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant β2-β3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site.

A mitochondrial EglN1-AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress.

Mitochondria play essential roles in cancer cell adaptation to hypoxia, but the underlying mechanisms remain elusive. Through mitochondrial proteomic profiling, we here find that the prolyl hydroxylase EglN1 (PHD2) accumulates on mitochondria under hypoxia. EglN1 substrate-binding region in the β2β3 loop is responsible for its mitochondrial translocation and contributes to breast tumor growth. Furthermore, we identify AMP-activated protein kinase alpha (AMPKα) as an EglN1 substrate on mitochondria. The EglN1-AMPKα interaction is essential for their mutual mitochondrial translocation. After EglN1 prolyl-hydroxylates AMPKα under normoxia, they rapidly dissociate following prolyl-hydroxylation, leading to their immediate release from mitochondria. In contrast, hypoxia results in constant EglN1-AMPKα interaction and their accumulation on mitochondria, leading to the formation of a Ca2+ /calmodulin-dependent protein kinase 2 (CaMKK2)-EglN1-AMPKα complex to activate AMPKα phosphorylation, ensuring metabolic homeostasis and breast tumor growth. Our findings identify EglN1 as an oxygen-sensitive metabolic checkpoint signaling hypoxic stress to mitochondria through its β2β3 loop region, suggesting a potential therapeutic target for breast cancer.

Interaction of Solanum tuberosum L. translation initiation factors eIF4E with potato virus Y VPg: Apprehend and avoid

Potato virus Y (PVY) is one of the most dangerous agricultural pathogens that causes substantial harm to vegetative propagated crops, such as potatoes (Solanum tuberosum L.). A necessary condition for PVY infection is an interaction between the plant cap-binding translation initiation factors eIF4E and a viral protein VPg, which mimics the cap-structure. In this study, we identified the point mutations in potato eIF4E1 and eIF4E2 that disrupt VPg binding while preserving the functional activity. For the structural interpretation of the obtained results, molecular models of all the studied forms of eIF4E1 and eIF4E2 were constructed and analyzed via molecular dynamics. The results of molecular dynamics simulations corresponds to the biochemical results and suggests that the β1β2 loop plays a key role in the stabilization of both eIF4E–cap and eIF4E–VPg complexes.

Cefiderocol activity is compromised by acquired extended-spectrum oxacillinases in Pseudomonas aeruginosa

Cefiderocol has an excellent in vitro activity on clinical strains of Pseudomonas aeruginosa (P. aeruginosa). However, the resistance of some isolates has been associated with the production of some β-lactamases. Whether some acquired extended-spectrum oxacillinases (ES-OXA) common in this species may compromise the susceptibility of P. aeruginosa to cefiderocol has not been evaluated so far. Eighteen genes encoding OXA belonging to the major subgroups identified in P. aeruginosa OXA-1 (n=3);-2 (n=5);-10 (n=8), and-46 (n=2) were cloned into pUCP24 shuttle vector and transferred into reference strain PAO1. Although production of the OXA-1 subgroup enzymes did not alter cefiderocol MICs, the β-lactamases of OXA-2, OXA-46, and four variants of the OXA-10 subgroup resulted in an 8-fold to 32-fold decrease in susceptibility in the PAO1 background. Interestingly, point mutations Ala149Pro and Asp150Gly in OXA-2 subgroup, Trp154Cys and Gly157Asp in OXA-10 subgroup (all located in the Ω loop), and the duplication of a Thr206 and a Gly207 in the β5-β6 loop of OXA-10 subgroup were related to decreased susceptibility to cefiderocol. We also showed that some ES-OXA, including the most frequent ES-OXA in P. aeruginosa strains, OXA-19 (derived from OXA-10 subgroup), significantly compromised activity of cefiderocol in addition to ceftazidime, ceftolozane/tazobactam, and ceftazidime/avibactam in clinical strains. This work shows that several ES-OXA have a significant effect on cefiderocol susceptibility. Of concern are the Trp154Cys and Gly157Asp mutations that occur in some of these β-lactamases, as they are associated with a decreased activity of the most recent cephalosporins introduced to combat P. aeruginosa infections.

Nonlocal Effects of Antibiotic-Resistance-Causing Mutations Reveal an Alternative Region for Targeting on FtsW-Penicillin-Binding Protein 3 Complex of Haemophilus influenzae.

Currently prescribed antibiotics target the catalytic sites of wild-type bacterial proteins; however, bacteria adopt mutations at this site, eventually leading to the emergence of resistance. Therefore, the identification of alternative drug binding sites is crucial, which requires knowledge of the dynamics of the mutant protein. Here, we set out to investigate the impact of a high-resistance-causing triple mutation (S385T + L389F + N526K) on the dynamics of a prioritized resistant pathogen, Haemophilus influenzae, using computational techniques. We studied penicillin-binding protein 3 (PBP3) and its complex with FtsW, which display resistance toward β-lactam antibiotics. We showed that mutations displayed local and nonlocal effects. In terms of the former, the orientation of the β-sheet, which surrounds the active site of PBP3, was impacted and the catalytic site was exposed to the periplasmic region. In addition, the flexibility of the β3-β4 loop, which modulates the catalysis of the enzyme, increased in the mutant FtsW-PBP3 complex. As for nonlocal effects, the dynamics of the pedestal domain (N-terminal periplasmic modulus (N-t)), i.e., the opening of the fork, was different between the wild-type and mutant enzymes. We showed the closed fork caused a greater number of residues to participate in the hypothesized allosteric communication network connecting N-t to the transpeptidase domain in the mutant enzyme. Finally, we demonstrated that the closed fork results in more favorable binding with β-lactam antibiotics, particularly cefixime, suggesting that small therapeutics that can stabilize the closed fork of mutant PBP3 may lead to the development of more effective molecules to combat resistant bacteria.

Two non-active site residues W165 and L166 prominently influence the beta-lactam hydrolytic ability of OXA-23 beta-lactamase.

Dissemination of class D OXA-type carbapenemases is one of the significant causes of beta-lactam resistance in Gram-negative bacteria. The amino acid residues present near the active site are involved in hydrolytic mechanism of class D carbapenemases, though it is not identified in OXA-23. Here, with the help of site-directed mutagenesis, we aimed to explicate the importance of the residues W165, L166 and V167 of the possible omega loop and residue D222 in the short β5-β6 loop on the activity of OXA-23. All the residues were substituted with alanine. The resultant proteins were assayed for the changes in activity in E. coli cells and purified for in vitro activity, and stability assessment. E. coli cells harboring OXA-23_W165A and OXA-23_L166A, individually, exhibited a significant decrease in resistance towards beta-lactam antibiotics as compared to OXA-23. Further, purified OXA-23_W165A and OXA-23_L166A imparted about >4-fold decrease in catalytic efficiency and displayed reduced thermal stability as compared to OXA-23. Bocillin-FL binding assay revealed that W165A substitution results in improper N-carboxylation of K82, leading to deacylation deficient OXA-23. Therefore, we infer that the residue W165 maintains the integrity of N-carboxylated lysine (K82) of OXA-23 and the residue L166 might be responsible for properly orientating the antibiotic molecules.

Mutation of valine 53 at the interface between extracellular and transmembrane domains of the β2 principal subunit affects the GABAA receptor gating

GABAA receptors (gamma-aminobutyric acid type A receptors) are pentameric ligand-gated ion channels mediating inhibition in adult mammalian brains. Their static structure has been intensely studied in the past years but the underlying molecular activatory mechanisms remain obscure. The interface between extracellular and transmembrane domains has been recognized as a key player in the receptor gating. However, the role of the valine 53 in the β1-β2 loop of the principal subunit (β2) remains controversial showing differences compared to homologous residues in some cys-loop counterparts such as nAChR. To address the role of the β2V53 residue in the α1β2γ2L receptor gating, we performed high resolution macroscopic and single-channel recordings. To explore underlying molecular mechanisms a variety of substituting amino acids were investigated: Glutamate and Lysine (different electric charge), Alanine (aliphatic, larger than Valine) and Histidine (same residue as in homologous α1H55). We report that mutation of the β2V53 residue results in alterations of nearly all gating transitions including opening/closing, preactivation and desensitization. A dramatic gating impairment was observed for glutamate substitution (β2V53E) but β2V53K mutation had a weak effect. The impact of histidine substitution was also small while β2V53A markedly affected the receptor but to a smaller extent than β2V53E. Considering available structures in desensitized and bicuculline blocked shut states we propose that strongly detrimental effect of β2V53E mutation on receptor activation results from electrostatic interaction between the glutamate and β2K274 on the loop M2-M3 which stabilizes the receptor in the shut state. We conclude that β2V53 is strongly involved in mechanisms underlying the receptor gating.

Solution structure of the DNA binding domain of Arabidopsis transcription factor WRKY11

The Arabidopsis WRKY11 (AtWRKY11) protein is an important transcription factor involved in plant response to biotic and abiotic stresses. Its DNA-binding domain specifically binds to gene promoter regions harboring the W-box consensus motif. Herein we report the high-resolution structure of the AtWRKY11 DNA-binding domain (DBD) determined by solution NMR spectroscopy. The results show that AtWRKY11-DBD adopts an all-β fold comprising five β-strands packed in an antiparallel topology, stabilized by a zinc-finger motif. Structural comparison reveals that the long β1-β2 loop shows the highest structural variation from other available WRKY domain structures. Moreover, this loop was further found to contribute to the binding between AtWRKY11-DBD and W-box DNA. Our current study provides atomic-level structural basis for further understanding the structure-function relationship of plant WRKY proteins.

Canonical or noncanonical? Structural plasticity of serine protease-binding loops in Kunitz-STI protease inhibitors.

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz-STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz-STI inhibitors to characterize serine protease-binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease-binding loops and the inhibition mechanism, their location in the β-trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4-β5 loop, whereas serine protease-binding loops in inhibitors from other plants lie mainly in the β5-β6 and β9-β10 loops. In addition, we found that the β5-β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz-STI inhibitors are canonical inhibitors and proteins with protease-binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease-binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.