Structure and backbone dynamics of vanadate-bound PRL-3: comparison of 15N nuclear magnetic resonance relaxation profiles of free and vanadate-bound PRL-3.

Phosphatases of regenerating liver (PRLs) constitute a novel class of small, prenylated phosphatases with oncogenic activity. PRL-3 is particularly important in cancer metastasis and represents a potential therapeutic target. The flexibility of the WPD loop as well as the P-loop of protein tyrosine phosphatases is closely related to their catalytic activity. Using nuclear magnetic resonance spectroscopy, we studied the structure of vanadate-bound PRL-3, which was generated by addition of sodium orthovanadate to PRL-3. The WPD loop of free PRL-3 extended outside of the active site, forming an open conformation, whereas that of vanadate-bound PRL-3 was directed into the active site by a large movement, resulting in a closed conformation. We suggest that vanadate binding induced structural changes in the WPD loop, P-loop, helices α4-α6, and the polybasic region. Compared to free PRL-3, vanadate-bound PRL-3 has a longer α4 helix, where the catalytic R110 residue coordinates with vanadate in the active site. In addition, the hydrophobic cavity formed by helices α4-α6 with a depth of 14-15 Å can accommodate a farnesyl chain at the truncated prenylation motif of PRL-3, i.e., from R169 to M173. Conformational exchange data suggested that the WPD loop moves between open and closed conformations with a closing rate constant k(close) of 7 s(-1). This intrinsic loop flexibility of PRL-3 may be related to their catalytic rate and may play a role in substrate recognition.

Structure and flexibility of the thermophilic cold-shock protein of Thermus aquaticus.

The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein (Ta-Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta-Csp has a five-stranded ß-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76 °C) of Ta-Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the ß1-ß2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674 s(-1). The results imply that thermophilic Ta-Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts.

Relationship between structural flexibility and function in the C-terminal region of the heparin-binding domain of VEGF165.

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid ß (Aß) interactions without affecting other biological activities of VEGF or Aß [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.

Structural and dynamic features of cold-shock proteins of Listeria monocytogenes, a psychrophilic bacterium.

Cold-shock proteins (Csps), proteins expressed when the ambient temperature drops below the growth-supporting temperature, bind to single-stranded nucleic acids and act as RNA chaperones to regulate translation. Listeria monocytogenes is a psychrophilic food-borne pathogen that is problematic for the food industry. Structures of Csps from psychrophilic bacteria have not yet been studied. Despite dramatic differences in the thermostability of Csps of various thermophilic microorganisms, these proteins share a high degree of primary sequence homology and a high degree of three-dimensional structural similarity. Here, we investigated the structural and dynamic features as well as the thermostability of L. monocytogenes CspA (Lm-CspA). Lm-CspA has a five-stranded ß-barrel structure with hydrophobic core packing and two salt bridges. When heptathymidine (dT(7)) binds, values for the heteronuclear nuclear Overhauser effect and order parameters of residues in surface loop regions near nucleic acid binding sites increase dramatically. Moreover, Carr-Purcell-Meiboom-Gill experiments showed that slow motions observed for the nucleic acid binding residues K7, W8, F15, F27, and R56 disappeared in Lm-CspA-dT(7). Lm-CspA is less thermostable than mesophilic and thermophilic Csps, with a lower melting temperature (40 °C). The structural flexibility that accompanies longer surface loops and less hydrophobic core packing and a number of salt bridges and unfavorable electrostatic repulsion are likely key factors in the low thermostability of Lm-CspA. This implies that the large conformational flexibility of psychrophilic Lm-CspA, which more easily accommodates nucleic acids at low temperature, is required for RNA chaperone function under cold-shock conditions and for the cold adaptation of L. monocytogenes.

Clinical Validation of the QMAC-DST System for Testing the Drug Susceptibility of Mycobacterium tuberculosis to First- and Second-Line Drugs.

There is a high demand for novel approaches to counter the various challenges of conventional drug susceptibility testing (DST) for tuberculosis, the most prevalent infectious disease with significant global mortality. The QMAC-DST system was recently developed for rapid DST using image technology to track the growth of single cells of Mycobacterium tuberculosis (MTB). The purpose of this study was to clinically validate the QMAC-DST system compared to conventional DST. In total, 178 MTB isolates recovered from clinical specimens in Asan Medical Center in 2016 were tested by both QMAC-DST and absolute concentration methods using Lowenstein-Jensen media (LJ-DST). Among the isolates, 156 were subjected to DST using BACTEC MGIT 960 SIRE kits (BD, Sparks, MD, United States) (MGIT-DST). The susceptibility/resistance results obtained by QMAC-DST were read against 13 drugs after 7 days of incubation and compared with those of LJ-DST. Based on the gold standard LJ-DST, the agreement rates of QMAC-DST for all drugs were 97.8%, 97.9%, and 97.8% among susceptible, resistant, and total isolates, respectively, while the overall agreement of MGIT-DST tested for 156 isolates against first-line drugs was 95.5%. QMAC-DST showed the highest major error of 6.4% for rifampin, however, it could be corrected by a revised threshold of growth since false-resistant isolates showed grew only half than the true-resistant isolates. The rapid and accurate performance of QMAC-DST warrants ideal phenotypic DST for a wide range of first-line and second-line drugs.

Direct, rapid antimicrobial susceptibility test from positive blood cultures based on microscopic imaging analysis.

For the timely treatment of patients with infections in bloodstream and cerebrospinal fluid, a rapid antimicrobial susceptibility test (AST) is urgently needed. Here, we describe a direct and rapid antimicrobial susceptibility testing (dRAST) system, which can determine the antimicrobial susceptibility of bacteria from a positive blood culture bottle (PBCB) in six hours. The positive blood culture sample is directly mixed with agarose and inoculated into a micropatterned plastic microchip with lyophilized antibiotic agents. Using microscopic detection of bacterial colony formation in agarose, the total time to result from a PBCB for dRAST was only six hours for a wide range of bacterial concentrations in PBCBs. The results from the dRAST system were consistent with the results from a standard AST, broth microdilution test. In tests of clinical isolates (n = 206) composed of 16 Gram-negative species and seven Gram-positive species, the dRAST system was accurate compared to the standard broth microdilution test, with rates of 91.11% (2613/2868) categorical agreement, 6.69% (192/2868) minor error, 2.72% (50/1837) major error and 1.45% (13/896) very major error. Thus, the dRAST system can be used to rapidly identify appropriate antimicrobial agents for the treatment of blood stream infection (BSI) and antibiotic-resistant strain infections.

Binding model for eriodictyol to Jun-N terminal kinase and its anti-inflammatory signaling pathway.

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-α, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signal-regulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, 8.79 × 10(5) M(-1). Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK.