Biolayer Interferometry Experiments Research Articles

Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus.

Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Potential Candidate Molecule of Photosystem II Inhibitor Herbicide-Brassicanate A Sulfoxide.

Brassicanate A sulfoxide, a secondary metabolite of broccoli, exhibited the inhibition of weed growth, but its mechanism of action on weeds remains unclear. To elucidate the mechanism by which brassicanate A sulfoxide suppresses weeds, this study explores the interaction between brassicanate A sulfoxide and the photosystem II D1 protein through molecular docking and molecular dynamics simulations. This research demonstrates that brassicanate A sulfoxide interacts with the photosystem II D1 protein by forming hydrogen bonds with Phe-261 and His-214. The successful expression of the photosystem II D1 protein in an insect cell/baculovirus system validated the molecular docking and dynamics simulations. Biolayer interferometry experiments elucidated that the affinity constant of brassicanate A sulfoxide with photosystem II was 2.69 × 10-3 M, suggesting that brassicanate A sulfoxide can stably bind to the photosystem II D1 protein. The findings of this study contribute to the understanding of the mode of action of brassicanate A sulfoxide and also aid in the development of natural-product-based photosynthesis-inhibiting herbicides.

Phage display assisted discovery of a pH-dependent anti-α-cobratoxin antibody from a natural variable domain library.

RecyclingIgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. As a result, an antibody was discovered that exhibits a 7-fold higher off-rate at pH 5.5 than pH 7.4 in bio-layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH-dependent binding to a histidine-devoid antigen mutant. As such, the results demonstrate that pH-dependent antigen-antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH-dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.

Widespread impact of immunoglobulin V-gene allelic polymorphisms on antibody reactivity

The ability of the human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V-gene allelic polymorphisms. However, previous studies have provided only limited examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many V-gene allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiments further demonstrate that paratope allelic polymorphisms on both heavy and light chains often abolish antibody binding. We also illustrate the importance of minor V-gene allelic polymorphisms with low frequency in several broadly neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Overall, this study not only highlights the pervasive impact of V-gene allelic polymorphisms on antibody binding but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.

An Experimental Framework for Developing Point-of-Need Biosensors: Connecting Bio-Layer Interferometry and Electrochemical Impedance Spectroscopy.

Biolayer interferometry (BLI) is a well-established laboratory technique for studying biomolecular interactions important for applications such as drug development. Currently, there are interesting opportunities for expanding the use of BLI in other fields, including the development of rapid diagnostic tools. To date, there are no detailed frameworks for implementing BLI in target-recognition studies that are pivotal for developing point-of-need biosensors. Here, we attempt to bridge these domains by providing a framework that connects output(s) of molecular interaction studies with key performance indicators used in the development of point-of-need biosensors. First, we briefly review the governing theory for protein-ligand interactions, and we then summarize the approach for real-time kinetic quantification using various techniques. The 2020 PRISMA guideline was used for all governing theory reviews and meta-analyses. Using the information from the meta-analysis, we introduce an experimental framework for connecting outcomes from BLI experiments (KD, kon, koff) with electrochemical (capacitive) biosensor design. As a first step in the development of a larger framework, we specifically focus on mapping BLI outcomes to five biosensor key performance indicators (sensitivity, selectivity, response time, hysteresis, operating range). The applicability of our framework was demonstrated in a study of case based on published literature related to SARS-CoV-2 spike protein to show the development of a capacitive biosensor based on truncated angiotensin-converting enzyme 2 (ACE2) as the receptor. The case study focuses on non-specific binding and selectivity as research goals. The proposed framework proved to be an important first step toward modeling/simulation efforts that map molecular interactions to sensor design.

Folding state as the Decision Maker in Detecting Misfolded Proteins by Hsp70

Maintaining cellular proteostasis is challenging due to the constantly changing and crowded cellular environment. Mutations, transcriptional and translational errors, and chemical and pathological stresses in a cell lead to protein misfolding and eventual formation of insoluble toxic aggregates. These aggregates typically result in loss of native protein function and are often associated with conformational diseases such as Alzheimer's, Parkinson's, Huntington's, and various types of cancer. The cellular protein quality control systems are essential for creating a stable cellular environment by resolving the misfolded proteins to uphold a stable proteome. The triage decision for a misfolded protein is regulated by Heat shock protein 70 (Hsp70), the central hub of protein quality control machinery, along with the E3 ubiquitin ligase C‐terminus of Hsc70‐interacting protein (CHIP). Hsp70 itself helps misfolded “client” proteins to refold and directs protein clients to downstream refolding pathways, whereas Hsp70 interaction with CHIP can lead to CHIP‐mediated ubiquitination, ultimately directing the proteins to proteasomal degradation. Understanding the molecular mechanisms that enable the Hsp70/CHIP complex to triage client proteins would allow for the identification of new avenues of treatment for the above‐mentioned diseases. With an objective to illustrate how the varying degrees of folding in a model client protein dictate the interaction between Hsp70 and the client, we have designed a spectrum of client proteins we named folding sensor TPRs (FSTPRs). We initially hypothesized that the more unfolded the client higher the affinity towards Hsp70. Circular dichroism (CD) spectroscopy was used to assess the folding states of each FSTPR variant, and biolayer interferometry (BLI) was utilized to quantify the affinities of each FSTPR towards the Hsp70 substrate‐binding domain (SBD). During our BLI experiments, we observed a range of threshold unfolding percentages that correspond to the highest affinity. Above and below the threshold range, the affinity decreased. This behavior was recapitulated with a disulfide‐locked FSTPR that is folded in the oxidized state and predominantly unfolded in the reduced state. Even though support from further data is necessary, these studies provide us preliminary insight into how Hsp70 interacts with a misfolded client and the role played by folding state in dictating Hsp70/client interactions.

Structural insights into DNA binding domain of vancomycin-resistance-associated response regulator in complex with its promoter DNA from Staphylococcus aureus.

In Staphylococcus aureus, vancomycin-resistance-associated response regulator (VraR) is a part of the VraSR two-component system, which is responsible for activating a cell wall-stress stimulon in response to an antibiotic that inhibits cell wall formation. Two VraR-binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N-terminal receiver domain (VraRN ) connected to a C-terminal DNA binding domain (VraRC ) with a flexible linker. Here, we present the crystal structure of VraRC alone and in complex with R1-DNA in 1.87- and 2.0-Å resolution, respectively. VraRC consisting of four α-helices forms a dimer when interacting with R1-DNA. In the VraRC -DNA complex structure, Mg2+ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg2+ to VraRC enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraRC with R1- and R2-DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic-resistant S. aureus via targeting VraR.

Suppressing Nonspecific Binding in Biolayer Interferometry Experiments for Weak Ligand-Analyte Interactions.

Quantitative analysis of protein–protein interactions (PPIs) using biolayer interferometry (BLI) requires effective suppression of nonspecific binding (NSB) between analytes and biosensors. In particular, the study of weak interactions (i.e., KD > 1 μM) requires high concentrations of analytes, which substantially increases NSB. However, there are only a few so-called NSB blockers compatible with biomolecules, which limits the use of BLI in the accurate analysis of weak interactions. The present study aims to identify a new NSB blocker for the quantitative analysis of weak PPIs using BLI. We find that saccharides, especially sucrose, are potent NSB blockers and demonstrate their compatibility with other blocking additives. We also demonstrate the effects of the new NSB blocker by characterizing the binding between nonstructural protein 1 of the influenza A virus and human phosphoinositide 3-kinase. We anticipate that the new NSB-blocking admixture will find broad applications in studying weak interactions using BLI.

A glycan gate controls opening of the SARS-CoV-2 spike protein

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded “down” to an exposed “up” state in order to bind the human ACE2 receptor and infect cells. While snapshots of the “up” and “down” states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 μs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia.

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines1-3. VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor4 (PF4)4; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis5, we found that the binding of anti-PF4 antibodies from patients with VITT (n=5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n=10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptorIIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis.

Q493K and Q498H substitutions in Spike promote adaptation of SARS-CoV-2 in mice.

BackgroundAn ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein .MethodsSARS-CoV-2 was passaged in BALB/c mice to obtain mouse-adapted virus strain. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments determined the binding affinity of mouse-adapted SARS-CoV-2 WBP-1 RBD to mouse ACE2 and human ACE2. Finally, we tested whether TLR7/8 agonist Resiquimod (R848) could also inhibit the replication of WBP-1 in the mouse model.FindingsThe mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, the study tentatively found that the TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge. Therefore, this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies.InterpretationWe found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies. This study also tentatively verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo.FundingThis research was funded by the National Key Research and Development Program of China (2020YFC0845600) and Emergency Science and Technology Project of Hubei Province (2020FCA046) and Robert A. Welch Foundation (C-1565).

Toll-Like Receptor Agonist R848 Protects Mice from Lethal SARS-CoV-2 Infections

Background: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Methods: We generated a mouse-adapted strain SARS-CoV-2 by serial passages in the lung of BALB/c mice. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments demonstrated that two mutations in RBD significantly increased its binding affinity towards mouse ACE2. Significantly, TLR7/8 agonist Resiquimod block SARS-CoV-2 in vitro and in vivo. Findings: We adapted a wild-type SARS-CoV-2 by serial passages in the lung of BALB/c mice. The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, The TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge, demonstrating this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. Interpretation: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies, especially in determining the immunopathological consequences of any intervention. This study also verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo.Funding Statement: This research was funded by Emergency Science and Technology Project of Hubei Province（2020FCA046）and Independent Science and Technology Innovation Fund of Huazhong Agricultural University in 2020 (2662020PY002).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The animal experiments were approved by the Research Ethics Committee, Huazhong Agricultural University, Hubei, China (HZAUMO-2020-0007). All the animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the Research Ethics Committee, Huazhong Agricultural University, Hubei, China.

Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike’s receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the “down” state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.

In vitro activity and In vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD target.

Staphylococcus xylosus (S. xylosus) is a type of coagulase-negative Staphylococcus, which was previously considered as non-pathogenic. However, recent studies have linked it with cases of mastitis in cows. Isoliquiritigenin (ISL) is a bioactive compound with pharmacological functions including antibacterial activity. In this study, we evaluated the effect of ISL on S. xylosus in vitro and in vivo. The MIC of ISL against S. xylosus was 80 μg/mL. It was observed that sub-MICs of ISL (1/2MIC, 1/4MIC, 1/8MIC) significantly inhibited the formation of S. xylosus biofilm in vitro. Previous studies have observed that inhibiting imidazole glycerol phosphate dehydratase (IGPD) concomitantly inhibited biofilm formation in S. xylosus. So, we designed experiments to target the formation of IGPD or inhibits its activities in S. xylosus ATCC 700404. The results indicated that the activity of IGPD and its histidine content decreased significantly under 1/2 MIC (40 μg/mL) ISL, and the expression of IGPD gene (hisB) and IGPD protein was significantly down-regulated. Furthermore, Bio-layer interferometry experiments showed that ISL directly interacted with IGPD protein (with strong affinity; KD = 234 μM). In addition, molecular docking was used to predict the binding mode of ISL and IGPD. In vivo tests revealed that, ISL significantly reduced TNF-α and IL-6 levels, mitigated the destruction of the mammary glands and reversed the production of inflammatory cells in mice. The results of the study suggest that, ISL may inhibit S. xylosus growth by acting on IGPD, which can be used as a target protein to treat infections caused by S. xylosus.

A Novel Heterozygous Missense Variant (c.667G>T;p.Gly223Cys) in USH1C That Interferes With Cadherin-Related 23 and Harmonin Interaction Causes Autosomal Dominant Nonsyndromic Hearing Loss.

BackgroundPathogenic variants of USH1C, encoding a PDZ-domain-containing protein called harmonin, have been known to cause autosomal recessive syndromic or nonsyndromic hearing loss (NSHL). We identified a causative gene in a large Korean family with NSHL showing a typical pattern of autosomal dominant (AD) inheritance.MethodsExome sequencing was performed for five affected and three unaffected individuals in this family. Following identification of a candidate gene variant, segregation analysis and functional studies, including circular dichroism and biolayer interferometry experiments, were performed.ResultsA novel USH1C heterozygous missense variant (c.667G>T;p.Gly223Cys) was shown to segregate with the NSHL phenotype in this family. This variant affects an amino acid residue located in the highly conserved carboxylate-binding loop of the harmonin PDZ2 domain and is predicted to disturb the interaction with cadherin-related 23 (cdh23). The affinity of the variant PDZ2 domain for a biotinylated synthetic peptide containing the PDZ-binding motif of cdh23 was approximately 16-fold lower than that of the wild-type PDZ2 domain and that this inaccessibility of the binding site was caused by a conformational change in the variant PDZ2 domain.ConclusionsA heterozygous variant of USH1C that interferes with the interaction between cdh23 and harmonin causes novel AD-NSHL.

Biophysical and functional characterizations of recombinant RimI acetyltransferase from Mycobacterium tuberculosis.

Nα-acetylation is a universal protein modification related to a wide range of physiological processes in eukaryotes and prokaryotes. RimI, an Nα-acetyltransferase in Mycobacterium tuberculosis, is responsible for the acetylation of the α-amino group of the N-terminal residue in the ribosomal protein S18. Despite growing evidence that protein acetylation may be correlated with the pathogenesis of tuberculosis, no structural information is yet available for mechanistically understanding the MtRimI acetylation. To enable structural studies for MtRimI, we constructed a serial of recombinant MtRimI proteins and assessed their biochemical properties. We then chose an optimal construct MtRimIC21A4-153 and expressed and purified the truncated high-quality protein for further biophysical and functional characterizations. The 2D 1H-15N heteronuclear single quantum coherence spectrum of MtRimIC21A4-153 exhibits wider chemical shift dispersion and favorable peak isolation, indicating that MtRimIC21A4-153 is amendable for further structural determination. Moreover, bio-layer interferometry experiments showed that MtRimIC21A4-153 possessed similar micromolar affinity to full-length MtRimI for binding the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg. Enzyme kinetic assays also exhibited that MtRimIC21A4-153 had almost identical enzymatic activity to MtRimI, indicating insignificant influence of the recombinant variations on enzymatic functions. Furthermore, binding sites of the peptide were predicted by molecular docking approach, suggesting that this substrate binds to MtRimI primarily through electrostatic and hydrogen bonding interactions. Our results lay a foundation for the further structural determination and dynamics detection of MtRimI.

Mortalin Caught in Action: Structures of the Nucleotide Binding Domain in the Apo, ADP‐bound and AMP+PPi‐bound States

The heat shock protein 70, Hsp70, chaperone family plays key roles in cellular homeostasis and stress response. Hsp70s not only regulate the folding of nascent proteins, but also prevent aggregation, promote disaggregation, and refold misfolded proteins. The mitochondrial Hsp70, Mortalin, functions in a variety of processes such as mitochondrial protein import and quality control, Fe‐S cluster biogenesis, mitochondrial homeostasis, and regulation of p53, an important tumor suppressor. Mortalin has been implicated in the regulation of apoptosis, cell stress response, neurodegenerative diseases, and has an anti‐apoptotic role in cancer cells. We have elucidated crystal structures of the nucleotide binding domain of human Mortalin in the apo, ADP‐, AMP and AMP states at 2.8 Å, 2.78 Å, and 1.75 Å resolutions, respectively. The crystal structure of Mortalin‐NBD binding to the modified ADP homolog N6propargyl‐ADP has also been determined at 1.49 Å. The overall structures and active site organizations are similar, with a few key differences in side chain positions. Specific residues in the nucleotide binding pocket, which are not conserved in other Hsp70‐family members, lead to lower nucleotide affinity and slower turnover relative to cytosolic Hsp70. ITC and biolayer interferometry experiments performed on the R126W and Y128C disease variants of Mortalin to determine ADP binding and interactions with an inter‐domain linker peptide elucidate how the mutations change Mortalin's functions and how these interactions differ between Mortalin, Hsp70, and Hsc70. ITC experiments have also been performed to test how the MortalinNBD can accommodate modified ADP nucleotides. This structural and binding data may also contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Streptococcuspneumoniae surface protein PfbA is a versatile multidomain and multiligand-binding adhesin employing different binding mechanisms.

Streptococcuspneumoniae, one of the major human respiratory pathogens, uses its repertoire of surface proteins to adhere to the epithelium of the nasopharynx and lungs leading to colonization. PfbA is a conserved surface protein of S.pneumoniae and helps the bacterium to colonize the host by recognizing the extracellular matrix (ECM) molecule fibronectin, as well as blood proteins like plasminogen and human serum albumin. The crystal structure of rPfbA150-607 revealed it to possess a beta-helical region similar to those of carbohydrate-active enzymes as well as a C-terminal segment that resembles the fibronectin-binding regions of fibronectin-binding proteins. To get more insight into the putative carbohydrate-binding property of PfbA and its binding to various host molecules, we generated three different constructs of PfbA and characterized them by ELISA, isothermal titration calorimetry and bio-layer interferometry experiments. Importantly, the isothermal titration calorimetry experiments revealed that PfbA binds to different saccharides. Further, ELISA and bio-layer interferometry experiments identified that (a) apart from fibronectin and plasminogen, the beta helix of PfbA also binds to other ECM molecules (b) lysines are not responsible for PfbA's binding to plasminogen, (c) in comparison with native fibrinogen, deglycosylated-fibrinogen exhibits reduced binding affinity towards PfbA implying the importance of sugar molecule-PfbA interaction and (d) the C-terminal region of PfbA binds exclusively to the N-terminal F1 modules of fibronectin. Thus, the results of this study show PfbA to be a versatile multidomain and multiligand-binding protein employing different binding mechanisms. These results could be useful for structure-based designing of inhibitors against PfbA.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.