Search

Background: Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease in women, while the oncogenic mechanisms which contribute to TNBC development, progression, and metastasis are poorly investigated and therefore warrant the critical need to identify novel oncogenic signalings and develop new therapeutic targets. Results: We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α protein level and activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggesting that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles in controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC. Conclusion: ZHX2 has been highlighted as one critical hypoxia-related factors regulator contributing to triple-negative breast cancer in this work, which has enriched the upstream regulatory network of HIF-1α signaling. Moreover, identification of key residuals determining biological function of ZHX2 provides novel approaches for treating TNBC via targeting the hypoxia pathway. Citation Format: Chengheng Liao, Wentong Fang, Rachel Shi, Jeremy Simon, Travis Ptacek, Giada Zurlo, Youqiong Ye, Leng Han, Cheng Fan, Lei Bao, Christopher Llynard Ortiz, Hong-Rui Lin, Ujjawal Manocha, Weibo Luo, Yan Peng, William Y Kim, Lee-Wei Yang, Qing Zhang. ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-23.

The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that it may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we find that centrosomes are highly accessible to soluble molecules with a Stokes radius of less than 5.8 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of this barrier is tightly regulated by branched actin filaments outside of centrosomes and it decreases during anaphase when branched actin temporally increases. The actin-based diffusion barrier gates microtubule nucleation by interfering with γ-tubulin ring complex recruitment. We propose that actin filaments spatiotemporally constrain protein complexes at centrosomes in a size-dependent manner.

Proteins sample an ensemble of conformers under physiological conditions, having access to a spectrum of modes of motions, also called intrinsic dynamics. These motions ensure the adaptation to various interactions in the cell, and largely assist in, if not determine, viable mechanisms of biological function. In recent years, machine learning frameworks have proven uniquely useful in structural biology, and recent studies further provide evidence to the utility and/or necessity of considering intrinsic dynamics for increasing their predictive ability. Efficient quantification of dynamics-based attributes by recently developed physics-based theories and models such as elastic network models provides a unique opportunity to generate data on dynamics for training ML models towards inferring mechanisms of protein function, assessing pathogenicity, or estimating binding affinities.

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In the present study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal “plugs” aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homomeric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable heteromeric channels did not. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus cells and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that the centrosome may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we found that centrosomes were highly accessible to soluble molecules with a Stokes radius of ≤ 5.1 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of barriers was tightly regulated by branched actin filaments outside of centrosomes. Such barrier gated the microtubule nucleation. We propose that actin filaments spatiotemporally constrain the distribution of molecules at centrosomes in a size-dependent manner.Significance StatementCentrosome maintains its microenvironment without membrane. Whether the dense protein complexes outside centrosomes including pericentriolar matrix, microtubules, and branched actin filaments provide physical obstruction is unclear yet. We here established a series of new tools for quantitative evaluation of the diffusion rates of varisized soluble proteins in different sub-compartments of centrosomes. Our results demonstrated that branched actin filaments, but not pericentriolar matrix or microtubules, around centrosome have acted as a size-dependent diffusion barrier and physically constrain centrosome microtubule nucleation.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease, which warrants the critical need to identify new therapeutic targets. We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggested that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles on controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC.

Motivation Quaternary structure determination for transmembrane/soluble proteins requires a reliable computational protocol that leverages observed distance restraints and/or cyclic symmetry (Cn symmetry) found in most homo-oligomeric transmembrane proteins. Results We survey 118 X-ray crystallographically solved structures of homo-oligomeric transmembrane proteins (HoTPs) and find that ∼97% are Cn symmetric. Given the prevalence of Cn symmetric HoTPs and the benefits of incorporating geometry restraints in aiding quaternary structure determination, we introduce two new filters, the distance-restraints (DR) and the Symmetry-Imposed Packing (SIP) filters. SIP relies on a new method that can rebuild the closest ideal Cn symmetric complex from docking poses containing a homo-dimer without prior knowledge of the number (n) of monomers. Using only the geometrical filter, SIP, near-native poses of 7 HoTPs in their monomeric states can be correctly identified in the top-10 for 71% of all cases, or 29% among 31 HoTP structures obtained through homology modeling, while ZDOCK alone returns 14 and 3%, respectively. When the n is given, the optional n-mer filter is applied with SIP and returns the near-native poses for 76% of the test set within the top-10, outperforming M-ZDOCK’s 55% and Sam’s 47%. While applying only SIP to three HoTPs that comes with distance restraints, we found the near-native poses were ranked 1st, 1st and 10th among 54 000 possible decoys. The results are further improved to 1st, 1st and 3rd when both DR and SIP filters are used. By applying only DR, a soluble system with distance restraints is recovered at the 1st-ranked pose. Availability and implementation https://github.com/capslockwizard/drsip. Supplementary information Supplementary data are available at Bioinformatics online.

In this study, we provide a time-dependent (td-) mechanical model, taking advantage of molecular dynamics (MD) simulations, quasiharmonic analysis of MD trajectories and td-linear response theories (td-LRT) to describe vibrational energy redistribution within the protein matrix. The theoretical description explains the observed biphasic responses of specific residues in myoglobin to CO-photolysis and photoexcitation on heme. The fast responses are found triggered by impulsive forces and propagated mainly by principal modes -1. The predicted fast responses for individual atoms are then used to study signal propagation within protein matrix and signals are found to propagate ∼ 8 times faster across helices (4076 m/s) than within the helices, suggesting the importance of tertiary packing in proteins’ sensitivity to external perturbations. We further develop a method to integrate multiple intramolecular signal pathways and discover frequent “communicators”. These communicators are found evolutionarily conserved including those distant from the heme.

MotivationDrug repurposing, where drugs originally approved to treat a disease are reused to treat other diseases, has received escalating attention especially in pandemic years. Structure-based drug design, integrating small molecular docking, molecular dynamic (MD) simulations and AI, has demonstrated its evidenced importance in streamlining new drug development as well as drug repurposing. To perform a sophisticated and fully automated drug screening using all the FDA drugs, intricate programming, accurate drug ranking methods and friendly user interface are very much needed.ResultsHere we introduce a new web server, DRDOCK, Drug Repurposing DOcking with Conformation-sampling and pose re-ranKing - refined by MD and statistical models, which integrates small molecular docking and molecular dynamic (MD) simulations for automatic drug screening of 2016 FDA-approved drugs over a user-submitted single-chained target protein. The drugs are ranked by a novel drug-ranking scheme using log-odds (LOD) scores, derived from feature distributions of true binders and decoys. Users can submit a selection of LOD-ranked poses for further MD-based binding affinity evaluation. We demonstrated that our platform can indeed recover one of the substrates for nsp16, a cap ribose 2′-O methyltransferase, and recommends that fluralaner, tegaserod and fenoterol could be repurposed for the COVID19 treatment with the latter two being confirmed in SARS-CoV2 suppression experiments. All the sampled docking poses and trajectories can be 3D-viewed and played via our web interface. This platform shall be easy-to-use for general scientists and medicinal researchers to carry out drug repurposing within a couple of days which should add value to our timely responses to, particularly, emergent disease outbreaks.Availability and implementationDRDOCK can be freely accessed from https://dyn.life.nthu.edu.tw/drdock/. (Due to the hardware upgrade, the service is NOT available before 7/18, 2021)

Motivation Programmed ribosomal frameshifting (PRF) is widely used by viruses and bacteria to produce different proteins from a single mRNA template. How steric hindrance of a PRF-stimulatory mRNA structure transiently modifies the conformational dynamics of the ribosome, and thereby allows tRNA slippage, remains elusive. Results Here, we leverage linear response theories and resolution-exchanged simulations to construct a structural/dynamics model that connects and rationalizes existing structural, single-molecule and mutagenesis data by resolution-exchanged structural modelling and simulations. Our combined theoretical techniques provide a temporal and spatial description of PRF with unprecedented mechanistic details. We discover that ribosomal unfolding of the PRF-stimulating pseudoknot exerts resistant forces on the mRNA entrance of the ribosome, and thereby drives 30S subunit rolling. Such motion distorts tRNAs, leads to tRNA slippage, and in turn serves as a delicate control of cis-element’s unwinding forces over PRF. Availability and implementation All the simulation scripts and computational implementations of our methods/analyses (including linear response theory) are included in the bioStructureM suite, provided through GitHub at https://github.com/Yuan-Yu/bioStructureM. Supplementary information Supplementary data are available at Bioinformatics online.

DynOmics (dynomics.pitt.edu) is a portal developed to leverage rapidly growing structural proteomics data by efficiently and accurately evaluating the dynamics of structurally resolved systems, from individual molecules to large complexes and assemblies, in the context of their physiological environment. At the core of the portal is a newly developed server, ENM 1.0, which permits users to efficiently generate information on the collective dynamics of any structure in PDB format, user-uploaded or database-retrieved. ENM 1.0 integrates two widely used elastic network models (ENMs)—the Gaussian Network Model (GNM) and the Anisotropic Network Model (ANM), extended to take account of molecular environment. It enables users to assess potentially functional sites, signal transduction or allosteric communication mechanisms, and protein–protein and protein–DNA interaction poses, in addition to delivering ensembles of accessible conformers reconstructed at atomic details based on the global modes of motions predicted by the ANM. The ‘environment’ is defined in a flexible manner, from lipid bilayer and crystal contacts, to substrate or ligands bound to a protein, or surrounding subunits in a multimeric structure or assembly. User-friendly interactive features permit users to easily visualize how the environment alter the intrinsic dynamics of the query systems. ENM 1.0 can be accessed at http://enm.pitt.edu/ or http://dyn.life.nthu.edu.tw/oENM/.

Structure-encoded conformational dynamics are crucial for biomolecular functions. However, there is insufficient evidence to support the notion that dynamics play a role in guiding protein-nucleic acid interactions. Here, we show that protein-DNA docking orientation is a function of protein intrinsic dynamics, but the binding site itself does not display unique patterns in the examined spectrum of motions. This revelation is made possible by a novel technique that locates "dynamics interfaces" in proteins across which protein parts are anticorrelated in their slowest dynamics. A striking statistic is that such interfaces intersect the DNA in 97% of the 104 examined cases. These findings were then used to screen decoys generated by rigid-body docking of DNA molecules onto DNA-binding proteins. Using our method, the chance to discern near-native poses from non-native decoys increased by 2.5- and 1.6-fold, as compared to a random guess and methods based on surface complementarity, respectively. Hence, dynamically allowed protein-DNA docking orientations can work as new filters to cull and rerank docking poses and therefore enhance the predictability of DNA-binding sites that themselves do not have distinct dynamics features. Computer software implementing the method can be accessed via http://dyn.life.nthu.edu.tw/IDD/DNA.htm .

To ensure a physicochemically desired sequence motif to adapt a specific type of secondary structures, we compile an α-helix database allowing complicate search patterns to facilitate a data-driven design of therapeutic peptides. Nearly 1.7 million helical peptides in >130 thousand proteins are extracted along with their interacting partners from the protein data bank (PDB). The sequences of the peptides are indexed with patterns and gaps and deposited in our Therapeutic Peptide Design dataBase (TP-DB). We here demonstrate its utility in three medicinal design cases. By our pattern-based search engine but not PHI-BLAST, we can identify a pathogenic protein, Helicobacter pylori neutrophil-activating protein (HP-NAP), a virulence factor of H. pylori, which contains a motif DYKYLE that belongs to the affinity determinant motif DYKXX[DE] of the FLAG-tag and can be recognized by the anti-FLAG M2 antibody. By doing so, the known purification-tag-specific antibody is repurposed into a diagnostic kit for H. pylori. Also by leveraging TP-DB, we discovered a stretch of helical peptide matching the potent membrane-insertion pattern WXXWXXW, elucidated by MD simulations. The newly synthesized peptide has a better minimal inhibitory concentration (MIC) and much lower cytotoxicity against Candida albicans (fungus) than that of previously characterized homologous antimicrobial peptides. In a similar vein, taking the discontinued anchoring residues in the helix-helix interaction interface as the search pattern, TP-DB returns several helical peptides as potential tumor suppressors of hepatocellular carcinoma (HCC) whose helicity and binding affinity were examined by MD simulations. Taken together, we believe that TP-DB and its pattern-based search engine provide a new opportunity for a (secondary-)structure-based design of peptide drugs and diagnostic kits for pathogens without inferring evolutionary homology between sequences sharing the same pattern. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/.

Motivation Quaternary structure determination for proteins is difficult especially for transmembrane proteins. Even if the monomeric constituents of complexes have been experimentally resolved, computational prediction of quaternary structures is a challenging task particularly for higher order complexes. It is essential to have a reliable computational protocol to predict quaternary structures of both transmembrane and soluble proteins leveraging experimentally determined distance restraints and/or cyclic symmetry (Cn symmetry) found in most homo-oligomeric transmembrane proteins. Results We survey 115 X-ray crystallographically solved structures of homo-oligomeric transmembrane proteins (HoTPs) to discover that 90% of them are Cn symmetric. Given the prevalence of Cn symmetric HoTPs and the benefits of incorporating geometry restraints in aiding quaternary structure determination, we introduce two new filters, the distance-restraints (DR) filter and the Symmetry-Imposed Packing (SIP) filter which takes advantage of the statistically derived tilt angle cutoff and the Cn symmetry of HoTPs without prior knowledge of the number (“n”) of monomers. Using only the geometrical filter, SIP, near-native poses of the 115 HoTPs can be correctly identified in the top-5 for 52% of all cases, or 49% among the HoTPs having an n >2 (~60% of the dataset), while ZDOCK alone returns 41% and 24%, respectively. Applying only SIP to three HoTPs with distance restraints, the near-native poses for two HoTPs are ranked 1st and the other 7th among 54,000 possible decoys. With both filters, the two remain 1st while the other improved to 2nd. While a soluble system with distance restraints is recovered at the 1st-ranked pose by applying only DR. Availability and Implementation https://github.com/capslockwizard/drsip Supplementary information Supplementary methods and results are available.

Table S1. Minimal inhibitory concentration breakpoints for Enterobacteriaceae. (DOCX 16 kb)

We provide evidence supporting that protein–protein and protein–ligand docking poses are functions of protein shape and intrinsic dynamics. Over sets of 68 protein–protein complexes and 240 nonhomologous enzymes, we recognize common predispositions for binding sites to have minimal vibrations and angular momenta, while two interacting proteins orient so as to maximize the angle between their rotation/bending axes (>65°). The findings are then used to define quantitative criteria to filter out docking decoys less likely to be the near-native poses; hence, the chances to find near-native hits can be doubled. With the novel approach to partition a protein into “domains” of robust but disparate intrinsic dynamics, 90% of catalytic residues in enzymes can be found within the first 50% of the residues closest to the interface of these dynamics domains. The results suggest an anisotropic rather than isotropic distribution of catalytic residues near the mass centers of enzymes.

Objective: The present study was designed to determine the neuroprotective effect of Abelmoschus esculentus L. on alloxan-induced diabetic neuropathy in rats.Methods: Diabetes was induced in rats with a single intraperitoneal injection of alloxan monohydrate (130 mg/kg b.w). The ethanol extract of A. esculentus L. at a dose of 100 and 200 mg/kg of body weight was administered at single dose per day to alloxan-induced diabetic rats for 21 days. The fasting blood glucose was screened in the intermittent on day 0, day 14, and day 21. Behavioral tests such as thermal hyperalgesia test and rotarod performance test were performed to assess the thermal sensitivity and muscle grip strength. At the end of the study period, experimental animals were sacrificed and sciatic nerve tissues were obtained for histopathological investigation.Results: Animals treated with A. esculentus L. extarct at a dose of 200 mg/kg of body weight significantly reduced (p

Human S100A9 (Calgranulin B) is a Ca(2+)-binding protein, from the S100 family, that often presents as a homodimer in myeloid cells. It becomes an important mediator during inflammation once calcium binds to its EF-hand motifs. Human RAGE protein (receptor for advanced glycation end products) is one of the target-proteins. RAGE binds to a hydrophobic surface on S100A9. Interactions between these proteins trigger signal transduction cascades, promoting cell growth, proliferation, and tumorigenesis. Here, we present the solution structure of mutant S100A9 (C3S) homodimer, determined by multi-dimensional NMR experiments. We further characterize the solution interactions between mS100A9 and the RAGE V domain via NMR spectroscopy. CHAPS is a zwitterionic and non-denaturing molecule widely used for protein solubilizing and stabilization. We found out that CHAPS and RAGE V domain would interact with mS100A9 by using (1)H-(15)N HSQC NMR titrations. Therefore, using the HADDOCK program, we superimpose two binary complex models mS100A9-RAGE V domain and mS100A9-CHAPS and demonstrate that CHAPS molecules could play a crucial role in blocking the interaction between mS100A9 and the RAGE V domain. WST-1 assay results also support the conclusion that CHAPS inhibits the bioactivity of mS100A9. This report will help to inform new drug development against cell proliferation.

Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1β) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1β signaling in vitro but can antagonize wild-type (WT) IL-1β. Crystal structures of T117A, E118A and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1β. Molecular dynamics simulations of IL-1β bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1β/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1β that are essential to immune responses and elucidate a mechanism by which IL-1β activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.

Motivation: A common practice in X-ray crystallographic structure refinement has been to model atomic displacements or thermal fluctuations as isotropic motions. Recent high-resolution data reveal, however, significant departures from isotropy, described by anisotropic displacement parameters (ADPs) modeled for individual atoms. Yet, ADPs are currently reported for a limited set of structures, only. Results: We present a comparative analysis of the experimentally reported ADPs and those theoretically predicted by the anisotropic network model (ANM) for a representative set of structures. The relative sizes of fluctuations along different directions are shown to agree well between experiments and theory, while the cross-correlations between the (x-, y-and z-) components of the fluctuations show considerable deviations. Secondary structure elements and protein cores exhibit more robust anisotropic characteristics compared to disordered or flexible regions. The deviations between experimental and theoretical data are comparable to those between sets of experimental ADPs reported for the same protein in different crystal forms. These results draw attention to the effects of crystal form and refinement procedure on experimental ADPs and highlight the potential utility of ANM calculations for consolidating experimental data or assessing ADPs in the absence of experimental data. Availability: The ANM server at http://www.ccbb.pitt.edu/anm is upgraded to permit users to compute and visualize the theoretical ADPs for any PDB structure, thus providing insights into the anisotropic motions intrinsically preferred by equilibrium structures. Contact: bahar@ccbb.pitt.edu Supplementary information: Two Supplementary Material files can be accessed at the journal website. The first presents the tabulated results from computations (Pearson correlations and KL distances with respect to experimental ADPs) reported for each of the 93 proteins in Set I (the averages over all proteins are presented above in Table 3). The second file consists of three sections: (A) detailed derivation of Equation (7), (B) analysis of the effect of ANM parameters on computed ADPs and identification of parameters that achieve optimal correlation with experiments and (C) description of the method for computing the tangential and radial components of equilibrium fluctuations.

Attempting different approaches to explore the best practice of optimizing mobile security and productivity is necessary to improve the Taiwan navy’s maneuverability and capability in the information age. This thesis uses a system engineering approach to research various mobile security technologies and applications that can provide the Taiwan navy with appropriate smartphone systems to develop a secure and productive smartphone incorporation plan. Having addressed system requirements, considered stakeholders’ concerns, and analyzed commercial off-the-shelf products, preliminary results have shown that Apple iOS is the most secure and productive system for smartphone incorporation in the Taiwan navy. Before rolling out a comprehensive incorporation plan, it is essential to evaluate the recommended technologies through a pilot program that simulates realistic naval activities to further correct deficiencies and determine the feasibility of the selected technologies. The intent of this research is to convince the Taiwan navy that secure smartphone incorporation is achievable through leveraging appropriate smartphone technologies and corresponding strategy, policy, and training. http://archive.org/details/thesystemenginee1094545888 Lieutenant, Taiwan Navy Approved for public release; distribution is unlimited.

With advances in structure genomics, it is now recognized that knowledge of structure alone is insufficient to understand and control the mechanisms of biomolecular function. Additional information in the form of dynamics is needed. As demonstrated in a large number of studies, the machinery of proteins and their complexes can be understood to a good approximation by adopting Gaussian (or elastic) network models (GNM) for simplified normal mode analyses. While this approximation lacks chemical details, it provides us with a means for assessing the collective motions of large structures/assemblies and perform a comparative analysis of a series of proteins, thus providing insights into the mechanical aspects of biomolecular dynamics. In this paper, we discuss recent applications of GNM to a series of enzymes as well as large structures such as the HK97 bacteriophage viral capsids. Understanding the dynamics of large protein structures can be computationally challenging. To this end, we introduce a new approach for building a hierarchical, reduced rank representation of the protein topology and consequently the fluctuation dynamics.

Motivation: The knowledge of protein structure is not sufficient for understanding and controlling its function. Function is a dynamic property. Although protein structural information has been rapidly accumulating in databases, little effort has been invested to date toward systematically characterizing protein dynamics. The recent success of analytical methods based on elastic network models, and in particular the Gaussian Network Model (GNM), permits us to perform a high-throughput analysis of the collective dynamics of proteins. Results: We computed the GNM dynamics for 20 058 structures from the Protein Data Bank, and generated information on the equilibrium dynamics at the level of individual residues. The results are stored on a web-based system called iGNM and configured so as to permit the users to visualize or download the results through a standard web browser using a simple search engine. Static and animated images for describing the conformational mobility of proteins over a broad range of normal modes are accessible, along with an online calculation engine available for newly deposited structures. A case study of the dynamics of 20 non-homologous hydrolases is presented to illustrate the utility of the iGNM database for identifying key residues that control the cooperative motions and revealing the connection between collective dynamics and catalytic activity. Availability: http://ignm.ccbb.pitt.edu/ Contact:[email protected] and [email protected]

The enzyme creatine amidinohydrolase is a clinically important enzyme used in the determination of creatinine in blood and urine. Continuous use biosensors are becoming more important in the clinical setting; however, long-use creatinine biosensors have not been commercialized due to the complexity of the three-enzyme creatinine biosensor and the lack of stability of its components. This paper, the second in a series of three, describes the immobilization and stabilization of creatine amidinohydrolase. Creatine amidinohydrolase modified with poly(ethylene glycol) activated with isocyanate retains significant activity after modification. The enzyme was successfully immobilized into hydrophilic polyurethanes using a reactive prepolymer strategy. The immobilized enzyme retained significant activity over a 30 day period at 37 °C and was irreversibly immobilized into the polymer. Despite being stabilized in the polymer, the enzyme remained highly sensitive to silver ions which were released from the amperometric electrodes. Computational analysis of the structure of the protein using the Gaussian network model suggests that the silver ions bind tightly to a cysteine residue preventing normal enzyme dynamics and catalysis.

The determination of creatinine levels in biological fluids is an increasingly important clinical requirement. Amperometric biosensors have been developed based on a three-enzyme system which converts creatinine to amperometrically measurable hydrogen peroxide. The development of the amperometric creatinine biosensor has been slow due the complexity of the three-enzyme system. This paper, the first of three, discusses the chemical modification of sarcosine oxidase and the immobilization and stabilization of this enzyme using polyurethane prepolymers. Sarcosine oxidase was completely inactivated after modification using poly(ethylene glycol) activated with isocyanate. The addition of a competitive inhibitor during enzyme modification was effective in protecting the enzyme from inactivation. Computational analysis of the structure of sarcosine oxidase suggests that there is a lysine in the active site that may be hyper-reactive. The enzyme was irreversibly immobilized using polyurethane prepolymers and retained significant activity. The enzyme’s half-life at 37 °C increased from seven days to more than 50 days after immobilization.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all- trans C 40 -octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s −1 ) was 100 times larger than the steady-state rate (0.02 s −1 ). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s −1 ) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.

S100B is a calcium sensing protein belonging to the S100 protein family with intracellular and extracellular roles. It is one of the EF hand homodimeric proteins, which is known to interact with various protein targets to regulate varied biological functions. Extracellular S100B has been recently reported to interact with FGF2 in a RAGE-independent manner. However, the recognition mechanism of S100B-FGF2 interaction at the molecular level remains unclear. In this study, the critical residues on S100B-FGF2 interface were mapped by combined information derived from NMR spectroscopy and site directed mutagenesis experiments. Utilizing NMR titration data, we generated the structural models of S100B-FGF2 complex from the computational docking program, HADDOCK which were further proved stable during 15ns unrestrained molecular dynamics (MD) simulations. Isothermal titration calorimetry studies indicated S100B interaction with FGF2 is an entropically favored process implying dominant role of hydrophobic contacts at the protein-protein interface. Residue level information of S100B interaction with FGF2 was useful to understand the varied target recognition ability of S100B and further explained its role in effecting extracellular signaling diversity. Mechanistic insights into the S100B-FGF2 complex interface and cell-based assay studies involving mutants led us to conclude the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (

The Lon protease is ubiquitous in nature. Its proteolytic activity is associated with diverse cellular functions ranging from maintaining proteostasis under normal and stress conditions to regulating cell metabolism. Although Lon was originally identified as an ATP-dependent protease with fused AAA+ (ATPases associated with diverse cellular activities) and protease domains, analyses have recently identified LonC as a class of Lon-like proteases with no intrinsic ATPase activity. In contrast to the canonical ATP-dependent Lon present in eukaryotic organelles and prokaryotes, LonC contains an AAA-like domain that lacks the conserved ATPase motifs. Moreover, the LonC AAA-like domain is inserted with a large domain predicted to be largely α-helical; intriguingly, this unique Lon-insertion domain (LID) was disordered in the recently determined full-length crystal structure of Meiothermus taiwanensis LonC (MtaLonC). Here, the crystal structure of the N-terminal AAA-like α/β subdomain of MtaLonC containing an intact LID, which forms a large α-helical hairpin protruding from the AAA-like domain, is reported. The structure of the LID is remarkably similar to the tentacle-like prong of the periplasmic chaperone Skp. It is shown that the LID of LonC is involved both in Skp-like chaperone activity and in recognition of unfolded protein substrates. The structure allows the construction of a complete model of LonC with six helical hairpin extensions defining a basket-like structure atop the AAA ring and encircling the entry portal to the barrel-like degradation chamber of Lon.

In this study, I present a new elastic network model, to our knowledge, that addresses insufficiencies of two conventional models—the Gaussian network model (GNM) and the anisotropic network model (ANM). It has been shown previously that the GNM is not rotation-invariant due to its energy, which penalizes rigid-body rotation (external rotation). As a result, GNM models are found contaminated with rigid-body rotation, especially in the most collective ones. A new model (EPIRM) is proposed to remove such external component in modes. The extracted internal motions result from a potential that penalizes interresidue stretching and rotation in a protein. The new model is shown to pertinently describe crystallographic temperature factors (B-factors) and protein open↔closed transitions. Also, the capability of separating internal and external motions in GNM slow modes permits reexamining important mechanochemical properties in enzyme active sites. The results suggest that catalytic residues stay closer to rigid-body rotation axes than their immediate backbone neighbors. I show that the cumulative density of states for EPIRM and ANM follow different power laws as functions of low-mode frequencies. When using a cutoff distance of 7.5 Å, The cumulative density of states of EPIRM scales faster than that of all-atom normal mode analysis and slower than that of simple lattices.

Atoms in proteins, viewed as interlinked hubs, communicate with each other in complying with governed physical forces. The deviations of their positions from the mean, known as fluctuations, are essential in mediating functionally relevant biological processes maintaining one's daily life. The coupled fluctuations between pairs of atoms, the fluctuation covariance, can be determined analytically by Normal Mode Analysis, or numerically by MD simulations. Our study considers how this network of atoms reacts in response to external perturbations. Effects of such perturbations are exemplified by ligand- or (another) protein-induced conformational changes as well as the appreciated structural distortion of crystalline structures from its solution conformers. We assume the response of the system has linear departure from the mean under small perturbations on the Hamiltonian at equilibrium state. The formulated linear response theories, either time-dependent or -independent [1], says that the positional change of a given atom i is the accumulative sum of fluctuation covariance ij, at unperturbed state, multiplied by the force exerted on atom j. The time-dependent response function determined from MD simulation of carbonmonoxy myoglobin is used to track time-dependent conformational changes upon photo-dissociation of CO. The consequently obtained understanding of perturbation propagation is compared with experimental results. Also, the structural distortion of X-ray-characterized ubiquitin from its solution conformer can be well explained by the induce-fit theory, in a wider sense, while population-shift does not account for such a deviation.[1] Ikeguchi M, Ueno J, Sato M, and Kidera A. (2005) Phys Rev Lett, 94, 078102.

Motivation: The Anisotropic Network Model (ANM) is a simple yet powerful model for normal mode analysis of proteins. Despite its broad use for exploring biomolecular collective motions, ANM has not been systematically evaluated to date. A lack of a convenient interface has been an additional obstacle for easy usage. Results: ANM has been evaluated on a large set of proteins to establish the optimal model parameters that achieve the highest correlation with experimental data and its limits of accuracy and applicability. Residue fluctuations in globular proteins are shown to be more accurately predicted than those in nonglobular proteins, and core residues are more accurately described than solvent-exposed ones. Significant improvement in agreement with experiments is observed with increase in the resolution of the examined structure. A new server for ANM calculations is presented, which offers flexible options for controlling model parameters and output formats, interactive animation of collective modes and advanced graphical features. Availability: ANM server () Contact: bahar@ccbb.pitt.edu

Function is a dynamic property closely related to the conformational mechanisms of the protein structure in its physiological environment. Efficient methods and tools for predicting collective motions at the molecular level are becoming increasingly important for understanding and controlling the function of target proteins. The molecular motions of proteins in different mode types are determined in the present study using the Gaussian network model (GNM). Based on GNM, we present a system that enables efficient query and visualization of protein dynamic behaviors such as mode shapes and 3D mobility structures.

Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.

The dnaX gene in Escherichia coli encodes comparable amounts of τ and γ subunits of DNA polymerase III by programmed ribosomal frameshifting (PRF). An early termination codon in the −1 frame of dnaX produces the γ subunit, which is a truncated form of the τ subunit. PRF is also involved in protein expression in many viruses, such as the Gag-Pol polyprotein in HIV. Thus, PRF provides an intrinsic regulating pathway at the translation level from the same mRNA.In the dnaX mRNA, an upstream internal Shine-Dalgarno (SD) sequence, which lies within the open reading frame and forms base-pairing with the 3’-end of 16S rRNA, together with a downstream stem-loop structure promote PRF on the slippery site. However, the detailed mechanisms for ribosome stalling and backtracking during PRF remain to be elucidated. Here, we combine coarse-grained elastic network model (ENM) and linear response theory to model the intrinsic and perturbed dynamics of the ribosome; the latter results from the formation of SD-helix and the forces exerted by stem-loop hindrance during translation. We hypothesize that these opposing forces for ribosome progression, albeit from different RNA elements, induce similar conformational changes for the ribosome, possibly in an allosteric fashion. These changes in the ribosome are expected to promote the observed PRF efficiency. Ultimately, we wish to provide the mechanochemical bases of PRF through computational modelling and validations by single-molecule force measurements.

Current study suggests that there exists an entropic restraint on protein-protein docking orientations. A physics-based, parameter-free approach has been developed to define intrinsic dynamic domains (IDDs) of proteins. Equilibrium dynamics of unbound protein pairs in 8 ‘difficult cases’ are sampled by Gaussian Network Model (GNM) and dimension-reduced Elastic Network Model (dr-ENM). IDDs are defined for proteins in accord with the slowest motions projected onto reduced dimensions. Domain planes and axes are thereby determined using Linear Discriminant Analysis (LDA) and principal components (PCs) of the position covariance of the unbound partners. The unbound partners are then best-fitted onto themselves in the complexes and the relative orientations of domain axes/planes of the paired proteins are examined. Our results show that proteins dock into each other with clear dynamic preferences in their orientations such that the binding planes of proteins cut through their docking partners, and also the bending axes of the docking pairs tend to be perpendicular to each other, implying proteins in the complexes are dynamically silent in the direction of their docking pairs’ vibrant movement. The dynamics-imposed restraints on protein docking orientations/locations shed light on entropic demands for proteins forming into complexes.

In this review, we summarize the progress on coarse-grained elastic network models (CG-ENMs) in the past decade. Theories were formulated to allow study of conformational dynamics in time/space frames of biological interest. Several highlighted models and their underlined hypotheses are introduced in physical depth. Important ENM offshoots, motivated to reproduce experimental data as well as to address the slow-mode-encoded configurational transitions, are also introduced. With the theoretical developments, computational cost is significantly reduced due to simplified potentials and coarse-grained schemes. Accumulating wealth of data suggest that ENMs agree equally well with experiment in describing equilibrium dynamics despite their distinct potentials and levels of coarse-graining. They however do differ in the slowest motional components that are essential to address large conformational changes of functional significance. The difference stems from the dissimilar curvatures of the harmonic energy wells described for each model. We also provide our views on the predictability of ‘open to close’ (open→close) transitions of biomolecules on the basis of conformational selection theory. Lastly, we address the limitations of the ENM formalism which are partially alleviated by the complementary CG-MD approach, to be introduced in the second paper of this two-part series.