Replica Exchange Molecular Dynamics Simulations Provide Insight into Substrate Recognition by Small Heat Shock Proteins

The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either “open” or “closed” based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency.     

Focus: Health Equity: Differences Risk Factors for Hypertension Among Elderly Woman in Rural and Urban Indonesia

Introduction: Hypertension is a major risk factor for cardiovascular disease. A high prevalence of hypertension is found in elderly women. Rural areas have different characteristics from urban areas. Therefore, it is necessary to identify risk factors for hypertension in rural and urban elderly women for optimal therapy management. Methods: This cross-sectional study was conducted in rural (Banyuwangi district) and urban (Surabaya city) areas, East Java, Indonesia. The study was carried out in 2015-2016 in women aged ≥45 years, residing in an area for ≥10 years, and willing to collect urine for 24 hours. Respondents consisted of 54 older adults from rural areas and 51 older adults from urban areas who actively participate in the integrated healthcare center for the elderly. The independent t-test and multivariate logistic regression were used to analyze the data. Results: The prevalence of hypertension in the rural area was 27.8% and in the urban area was 37.25%. The risk factors for hypertension in the urban area were urine sodium level (AOR=1.02, 95% CI=1.001-1.04, p-value=0.043), urine potassium level (AOR=0.88, 95% CI=0.78-0.999, p-value=0.022), and Body Mass Index (AOR=1.26, 95% CI=1.06-1.49, p-value=0.008). Meanwhile, the factor associated with hypertension in the rural area was age (AOR=1.08, 95% CI=1.003-1.16, p-value=0.042). Conclusion: The prevalence of hypertension in the urban area was higher than in the rural area. There were differences in risk factors for hypertension that occurred both in rural and urban areas. However, risk factors in both areas are equally important to overcome. Collaboration from multiple stakeholders and sectors is urgently needed, such as the Public Health Center, Integrated Assistance Post for Non-Communicable Diseases, and the local health office.     

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.     

Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.     

Flexible fitting of high-resolution x-ray structures into cryoelectron microscopy maps using biased molecular dynamics simulations

A methodology for flexible fitting of all-atom high-resolution structures into low-resolution cryoelectron microscopy (cryo-EM) maps is presented. Flexibility of the modeled structure is simulated by classical molecular dynamics and an additional effective potential is introduced to enhance the fitting process. The additional potential is proportional to the correlation coefficient between the experimental cryo-EM map and a synthetic map generated for an all-atom structure being fitted to the map. The additional forces are calculated as a gradient of the correlation coefficient. During the molecular dynamics simulations under the additional forces, the molecule undergoes a conformational transition that maximizes the correlation coefficient, which results in a high-accuracy fit of all-atom structure into a cryo-EM map. Using five test proteins that exhibit structural rearrangement during their biological activity, we demonstrate performance of our method. We also test our method on the experimental cryo-EM of elongation factor G and show that the model obtained is comparable to previous studies. In addition, we show that overfitting can be avoided by assessing the quality of the fitted model in terms of correlation coefficient and secondary structure preservation.     

A new mutation Rim3 resembling Re den is mapped close to retinoic acid receptor alpha (Rara) gene on mouse Chromosome 11

A new mouse mutation, recombination-induced mutation 3 (Rim3), arose spontaneously in our mouse facility. This mutation exhibits corneal opacity as well as abnormal skin and hair development resembling rex denuded (Re ^den ) and bareskin (Bsk). Large-scale linkage analysis with two kinds of intersubspecific backcrosses revealed that Rim3 is mapped to the distal portion of Chromosome (Chr) 11, in which Re ^den and Bsk have been located, and is very close to the retinoic acid receptor, alpha (Rara). The genes, keratin gene complex-1, acidic, gene 10, 12 (Krt1-10, 12), granulin (Grn), junctional plakoglobin (Jup) and Rara, all of which regulate growth and differentiation of epithelial cells, are genetically excluded as candidate genes for Rim3, but are clustered in the short segment on mouse Chr 11. Received: 3 July 1997 / Accepted: 26 August 1997     

Corn and kidney bean root endoplasmic reticulum vesicles reduce a fungal iron chelate,ferrirhodotorulic acid,in vitro

Summary Endoplasmic reticulum vesicles from both corn and kidney bean roots are capable of reducing ferrirhodotorulic acid, a fungal iron chelator, in vitro, using NADH as the reductant. In magnesium containing linear 15–45% sucrose density gradients, the activity was in a wide, high density band. The activity shifted in density to 1.07–1.08 when EDTA was included instead of magnesium. No plasma membrane reducing activity was found, even with iron deficient plants, using both NADH and NADPH as electron donors. It is speculated that the Eo value for ferrirhodotorulic acid, –0.36 V, may be too low for this chelate to be reduced by the iron deficiency induced iron chelate reductase found in the plasma membrane of nonpoaceous plants. Most of the activity was in the microsomal fraction rather than the soluble fraction.Abbreviations EDTA ethylene diamine tetraacetic acid - DTT dithiothreitol - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinyl polypyrrolidone - BPDS bathophenanthroline disulfonate - MES 2-(n-morpholino)ethanesulfonic acid