Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Structure-based functional identification of Helicobacter pylori HP0268 as a nuclease with both DNA nicking and RNase activities.

HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn(2+) and Mg(2+) ions, respectively, and decreased by Cu(2+) ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases.

Application of Solution NMR to Structural Studies on α-Helical Integral Membrane Proteins.

A large portion of proteins in living organisms are membrane proteins which play critical roles in the biology of the cell, from maintenance of the biological membrane integrity to communication of cells with their surroundings. To understand their mechanism of action, structural information is essential. Nevertheless, structure determination of transmembrane proteins is still a challenging area, even though recently the number of deposited structures of membrane proteins in the PDB has rapidly increased thanks to the efforts using X-ray crystallography, electron microscopy, and solid and solution nuclear magnetic resonance (NMR) technology. Among these technologies, solution NMR is a powerful tool for studying protein-protein, protein-ligand interactions and protein dynamics at a wide range of time scales as well as structure determination of membrane proteins. This review provides general and useful guideline for membrane protein sample preparation and the choice of membrane-mimetic media, which are the key step for successful structural analysis. Furthermore, this review provides an opportunity to look at recent applications of solution NMR to structural studies on α-helical membrane proteins through some success stories.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Dimerization of TRAF-interacting protein (TRAIP) regulates the mitotic progression.

The homo- or hetero-dimerization of proteins plays critical roles in the mitotic progression. The TRAF-interacting protein (TRAIP) is crucial in early mitotic progression and chromosome alignment defects in the metaphase. The TRAIP is a 469 amino acid protein, including the Really Interesting New Gene (RING), coiled-coil (CC), and leucine zipper (LZ) domain. In general, the CC or LZ domain containing proteins forms homo- or hetero-dimerization to achieve its activity. In this study, a number of TRAIP mutants were used to define the TRAIP molecular domains responsible for its homo-dimerization. A co-immunoprecipitation assay indicated that the TRAIP forms homo-dimerization through the CC domain. The cells, expressing the CC domain-deleted mutant that could not form a homo-dimer, increased the mitotic index and promoted mitotic progression.

The C-terminal domain of the transcriptional regulator BldD from Streptomyces coelicolor A3(2) constitutes a novel fold of winged-helix domains.

BldD regulates transcription of key developmental genes in Streptomyces coelicolor. While the N-terminal domain is responsible for both dimerization and DNA binding, the structural and functional roles of the C-terminal domain (CTD) remain largely unexplored. Here, the solution structure of the BldD-CTD shows a novel winged-helix domain fold not compatible with DNA binding, due to the negatively charged surface and presence of an additional helix. Meanwhile, a small elongated groove with conserved hydrophobic patches surrounded by charged residues suggests that the BldD-CTD could be involved in protein-protein interactions that provide transcriptional regulation.

Structures of inactive CRP species reveal the atomic details of the allosteric transition that discriminates cyclic nucleotide second messengers.

The prokaryotic global transcription factor CRP has been considered to be an ideal model for in-depth study of both the allostery of the protein and the differential utilization of the homologous cyclic nucleotide second messengers cAMP and cGMP. Here, atomic details from the crystal structures of two inactive CRP species, an apo form and a cGMP-bound form, in comparison with a known active conformation, the cAMP-CRP complex, provide macroscopic and microscopic insights into CRP allostery, which is coupled to specific discrimination between the two effectors. The cAMP-induced conformational transition, including dynamic fluctuations, can be driven by the fundamental folding forces that cause water-soluble globular proteins to construct an optimized hydrophobic core, including secondary-structure formation. The observed conformational asymmetries underlie a negative cooperativity in the sequential binding of cyclic nucleotides and a stepwise manner of binding with discrimination between the effector molecules. Additionally, the finding that cGMP, which is specifically recognized in a syn conformation, induces an inhibitory conformational change, rather than a null effect, on CRP supports the intriguing possibility that cGMP signalling could be widely utilized in prokaryotes, including in aggressive inhibition of CRP-like proteins.

Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

Structural characterization of de novo designed L5K5W model peptide isomers with potent antimicrobial and varied hemolytic activities.

In an effort to develop short antimicrobial peptides with simple amino acid compositions, we generated a series of undecapeptide isomers having the L(5)K(5)W formula. Amino acid sequences were designed to be perfectly amphipathic when folded into a helical conformation by converging leucines onto one side and lysines onto the other side of the helical axis. The single tryptophans, whose positions were varied in the primary structures, were located commonly at the critical amphipathic interface in the helical wheel projection. Helical conformations and the tryptophanyl environments of the 11 L(5)K(5)W peptides were confirmed and characterized by circular dichroism, fluorescence and nuclear magnetic resonance spectroscopy. All of the isomers exhibited a potent, broad-spectrum of antibacterial activity with just a slight variance in individual potency, whereas their hemolytic activities against human erythrocytes were significantly diversified. Interestingly, helical dispositions and fluorescence blue shifts of the peptides in aqueous trifluoroethanol solutions, rather than in detergent micelles, showed a marked linear correlation with their hemolytic potency. These results demonstrate that our de novo design strategy for amphipathic helical model peptides is effective for developing novel antimicrobial peptides and their hemolytic activities can be estimated in correlation with structural parameters.

Polyphenols Coordinated with Cu (II) in an Aqueous System Build Ion-Channel Coatings on Hair Surfaces.

Recently, developments in the field of cosmetics have led to a renewed interest in hair dyeing. However, damage to the hair during the dyeing process has increased hesitation in attempting hair dyeing. As a result, hair dyes with minimal side effects have been in constant demand, and are being developed. In this study, natural-extract polyphenols, pyrogallol, and gallic acid are coordinated by CuCl2 in a NaCl aqueous solution to form an oligomer, which creates an ion-channel coating on the hair surface to protect it. This work attempts to develop fast, simple, and damage-free hair-dye ingredients based on pyrogallol and gallic acid. The morphology and elements of polyphenols coated on hair are characterized. The results reveal that the hair is dyed with the polyphenol-based dye reagent successfully. Moreover, the thickness of the dyed hair continuously rises ten times after dyeing. The tensile strength of the dyed hair is also measured, showing an upward and downward trend. These results reflect the fact that pyrogallol and gallic acid are considered to be the essential and functional polyphenols, and can build ion blocks on hair, which can create new multifunctional coating materials.

Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

Coiled-coil structure mediated inhibition of the cytotoxic huntingtin amyloid fibrils by an IP3 receptor fragment.

Disruption of cellular homeostasis by the aggregation of polyglutamine (polyQ) in the huntingtin protein (Htt) leads Huntington's disease (HD). Effective drugs for treating HD have not been developed, as the molecular mechanism underlying HD pathogenesis remains unclear. To develop strategies for inhibiting HD pathogenesis, the intermolecular interaction of Htt with IP3 receptor 1 (IP3R1) was investigated. Peptide (termed ICT60) corresponding to a coiled-coil motif in the C-terminus of IP3R1 was designed. Several biophysical approaches revealed the strong and specific binding of ICT60 to the N-terminal part of HttEx1. ICT60 inhibited not only amyloid formation by HttEx1, but also the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1. The importance of coiled-coil structure was verified by charge-manipulated variants. The coiled-coil structures of ICT60-KK and -EE were partially and largely disrupted, respectively. ICT60 wild-type and -KK inhibited amyloid formation by HttEx1-46Q, whereas ICT60-EE did not block amyloidogenesis. Similarly, the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1-46Q were efficiently inhibited by ICT60 wild-type and ICT60-KK, but not by ICT60-EE. We propose a mechanical model explaining how an IP3 receptor-inspired molecule can modulate cytotoxic amyloid formation by Htt, providing a molecular basis for developing therapeutics to treat HD.

Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions.

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

Antimicrobial peptides for therapeutic applications: a review.

Antimicrobial peptides (AMPs) have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to conventional antibiotics. Although AMPs possess considerable benefits as new generation antibiotics, their clinical and commercial development still have some limitations, such as potential toxicity, susceptibility to proteases, and high cost of peptide production. In order to overcome those obstacles, extensive efforts have been carried out. For instance, unusual amino acids or peptido-mimetics are introduced to avoid the proteolytic degradation and the design of short peptides retaining antimicrobial activities is proposed as a solution for the cost issue. In this review, we focus on small peptides, especially those with less than twelve amino acids, and provide an overview of the relationships between their three-dimensional structures and antimicrobial activities. The efforts to develop highly active AMPs with shorter sequences are also described.

10-mer and 9-mer WALK Peptides with Both Antibacterial and Anti-Inflammatory Activities.

Natural antimicrobial peptides (AMPs) are multifunctional host defense peptides (HDPs) that are valuable for various therapeutic applications. In particular, natural and artificial AMPs with dual antibacterial immunomodulatory functions emerged as promising candidates for the development of therapeutic agents to treat infectious inflammation. In an effort to develop useful AMP variants with short lengths and simple amino acid composition, we devised a de novo design strategy to generate a series of model peptide isomer sequences, named WALK peptides, i.e., tryptophan (W)-containing amphipathic-helical (A) leucine (L)/lysine (K) peptides. Here, we generated two groups of WALK peptide isomers: W2L4K4 (WALK244.01~WALK244.10) and W2L4K3 (WALK243.01~WALK243.09). Most showed apparent antibacterial activities against both Gram-positive and Gram-negative bacteria at a concentration of approximately 4 µg/mL along with varied hemolytic activities against human red blood cells. In addition, some exhibited significant anti-inflammatory activities without any significant cytotoxicity in macrophages. Collectively, these results suggest that the two selected peptides, WALK244.04 and WALK243.04, showed promise for the development of antibacterial and anti-inflammatory agents.

Structural resemblance of the DNAJA-family protein, Tid1, to the DNAJB-family Hsp40.

The specific pair of heat shock protein 70 (Hsp70) and Hsp40 constitutes an essential molecular chaperone system involved in numerous cellular processes, including the proper folding/refolding and transport of proteins. Hsp40 family members are characterized by the presence of a conserved J-domain (JD) that functions as a co-chaperone of Hsp70. Tumorous imaginal disc 1 (Tid1) is a tumor suppressor protein belonging to the DNAJA3 subfamily of Hsp40 and functions as a co-chaperone of the mitochondrial Hsp70, mortalin. In this work, we performed nuclear magnetic resonance spectroscopy to determine the solution structure of JD and its interaction with the glycine/phenylalaninerich region (GF-motif) of human Tid1. Notably, Tid1-JD, whose conformation was consistent with that of the DNAJB1 JD, appeared to stably interact with its subsequent GF-motif region. Collectively with our sequence analysis, the present results demonstrate that the functional and regulatory mode of Tid1 resembles that of the DNAJB1 subfamily members rather than DNAJA1 or DNAJA2 subfamily proteins. Therefore, it is suggested that an allosteric interaction between mortalin and Tid1 is involved in the mitochondrial Hsp70/Hsp40 chaperone system. [BMB Reports 2022; 55(10): 488-493].

Crystallization and X-ray data collection of HP0902 from Helicobacter pylori 26695.

HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His(6) tag ((H6)HP0902) and the other with a C-terminal His(6) tag (HP0902(H6)). The (H6)HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902(H6) crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å.

Molecular Effects of Elongation Factor Ts and Trigger Factor on the Unfolding and Aggregation of Elongation Factor Tu Induced by the Prokaryotic Molecular Chaperone Hsp33.

Hsp33, a prokaryotic redox-regulated holding chaperone, has been recently identified to be able to exhibit an unfoldase and aggregase activity against elongation factor Tu (EF-Tu) in its reduced state. In this study, we investigated the effect of elongation factor Ts (EF-Ts) and trigger factor (TF) on Hsp33-mediated EF-Tu unfolding and aggregation using gel filtration, light scattering, circular dichroism, and isothermal titration calorimetry. We found that EF-Tu unfolding and subsequent aggregation induced by Hsp33 were evident even in its complex state with EF-Ts, which enhanced EF-Tu stability. In addition, although TF alone had no substantial effect on the stability of EF-Tu, it markedly amplified the Hsp33-mediated EF-Tu unfolding and aggregation. Collectively, the present results constitute the first example of synergistic unfoldase/aggregase activity of molecular chaperones and suggest that the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones to regulate protein biosynthesis in cells under stress conditions.

Structural Interplays in the Flexible N-Terminus and Scaffolding Domain of Human Membrane Protein Caveolin 3.

Caveolins are critical for the formation of caveolae, which are small invaginations of the plasma membrane involved in a variety of biological processes. Caveolin 3 (Cav3), one of three caveolin isoforms, is an integral membrane protein mainly expressed in muscle tissues. Although various human diseases associated with Cav3 have been reported, structural characterization of Cav3 in the membrane has not been investigated in enough depth to understand the structure-function relationship. Here, using solution NMR, we characterized membrane association, structural communications, and molecular dynamics of the monomeric Cav3 in detergent micelle environment, particularly focused on the whole N-terminal part that is composed of the flexible N-terminus and the scaffolding domain. The results revealed a complicated structural interplay of the individual segments composing the whole N-terminal part, including the pH-dependent helical region, signature motif-like region, signature motif, and scaffolding domain. Collectively, the present study provides novel structural insights into the whole N-terminal part of Cav3 that plays important biological roles in cellular processes and diseases. In particular, given that several disease-related mutations are located at the whole N-terminal part of Cav3, the sophisticated communications in the whole N-terminal segments are likely to have relevance to the molecular basis of Cav3-related disease.

Action mechanism and structural requirements of the antimicrobial peptides, gaegurins.

Gaegurins (GGNs) are a family of cationic, alpha-helical, antimicrobial peptides that were isolated from a Korean frog, Glandirana emeljanovi (formerly classified as Rana rugosa) and represent one of the structurally well-characterized groups. Among six gaegurins, gaegurin 4 (renamed herein esculentin-2EM), gaegurin 5 (brevinin-1EMa), and gaegurin 6 (brevinin-1EMb) have been investigated comprehensively in terms of structure-activity relationships. In this paper, we first suggest renaming of gaegurins according to a recently raised rule of systematic nomenclature. Then, the current understanding of gaegurins is reviewed by summarizing their structure-activity relationships. In particular competing arguments on gaegurins are synthetically inspected. Finally their action mechanism and structural requirements will be discussed.