scholarly journals Crystal structure of the sodium–proton antiporter NhaA dimer and new mechanistic insights

2014 ◽  
Vol 144 (6) ◽  
pp. 529-544 ◽  
Author(s):  
Chiara Lee ◽  
Shoko Yashiro ◽  
David L. Dotson ◽  
Povilas Uzdavinys ◽  
So Iwata ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Proton Transport ◽  
Salt Bridge ◽  
Sodium Concentration ◽  
Crystal Form ◽  
Ion Binding ◽  
Sodium Ions ◽  
The Stability ◽  
Dynamics Simulations

Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.

scholarly journals Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies

2019 ◽  
Vol 20 (4) ◽  
pp. 860 ◽  
Author(s):  
Joao Ramos ◽  
Jayaraman Muthukumaran ◽  
Filipe Freire ◽  
João Paquete-Ferreira ◽  
Ana Otrelo-Cardoso ◽  
...  
Keyword(s):  
Conformational Changes ◽  
In Silico ◽  
Nucleotide Polymorphisms ◽  
Ligand Interaction ◽  
Zinc Database ◽  
In Silico Studies ◽  
Anti Cancer ◽  
Binding Groove ◽  
The Stability ◽  
Dynamics Simulations

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

scholarly journals A two-domain elevator mechanism for sodium/proton antiport

2014 ◽  
Vol 70 (a1) ◽  
pp. C1048-C1048
Author(s):  
Chiara Lee ◽  
Hae Joo Kang ◽  
Christoph von Ballmoos ◽  
Simon Newstead ◽  
Povilas Uzdavinys ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Drug Targets ◽  
Thermus Thermophilus ◽  
Ion Binding ◽  
Dimerization Domain ◽  
Core Domain ◽  
Large Rotation ◽  
The Core ◽  
Dynamics Simulations

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 200against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy

2014 ◽  
Vol 70 (7) ◽  
pp. 1965-1976 ◽  
Author(s):  
Clorinda Lori ◽  
Alessandra Pasquo ◽  
Roberta Montanari ◽  
Davide Capelli ◽  
Valerio Consalvi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Partial Agonist ◽  
Salt Bridge ◽  
Van Der Waals Interactions ◽  
Structural Basis ◽  
Wild Type ◽  
Partial Lipodystrophy ◽  
X Ray ◽  
Familial Partial Lipodystrophy

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.

scholarly journals How well do force fields capture the strength of salt bridges in proteins?

2018 ◽  
Author(s):  
Mustapha Carab Ahmed ◽  
Elena Papaleo ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Force Fields ◽  
Salt Bridge ◽  
Salt Bridges ◽  
Enhanced Sampling ◽  
Important Interaction ◽  
Close Proximity ◽  
The Stability ◽  
Dynamics Simulations

AbstractSalt bridges form between pairs of ionisable residues in close proximity and are important interactions in proteins. While salt bridges are known to be important both for protein stability, recognition and regulation, we still do not have fully accurate predictive models to assess the energetic contributions of salt bridges. Molecular dynamics simulations is one technique that may be used study the complex relationship between structure, solvation and energetics of salt bridges, but the accuracy of such simulations depend on the force field used. We have used NMR data on the B1 domain of protein G (GB1) to benchmark molecular dynamics simulations. Using enhanced sampling simulations, we calculated the free energy of forming a salt bridge for three possible ionic interactions in GB1. The NMR experiments showed that these interactions are either not formed, or only very weakly formed, in solution. In contrast, we show that the stability of the salt bridges is slightly overestimated in simulations of GB1 using six commonly used combinations of force fields and water models. We therefore conclude that further work is needed to refine our ability to model quantitatively the stability of salt bridges through simulations, and that comparisons between experiments and simulations will play a crucial role in furthering our understanding of this important interaction.

scholarly journals Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

2020 ◽  
Author(s):  
Patrick Johé ◽  
Elmar Jaenicke ◽  
Hannes Neuweiler ◽  
Tanja Schirmeister ◽  
Christian Kersten ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Salt Bridge ◽  
Correlation Spectroscopy ◽  
African Trypanosomes ◽  
Lysosomal Cysteine Protease ◽  
Control Switch ◽  
Α Helix ◽  
Dynamics Simulations

AbstractRhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

scholarly journals Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin

2019 ◽  
Vol 116 (23) ◽  
pp. 11229-11234 ◽  
Author(s):  
Marte Innselset Flydal ◽  
Martín Alcorlo-Pagés ◽  
Fredrik Gullaksen Johannessen ◽  
Siseth Martínez-Caballero ◽  
Lars Skjærven ◽  
...  
Keyword(s):  
Phenylalanine Hydroxylase ◽  
Genetic Disorder ◽  
Interaction Network ◽  
Salt Bridge ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Pharmacological Chaperone ◽  
Key Enzyme ◽  
The Stability ◽  
Dynamics Simulations

Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4. BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII. Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4. Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations.

scholarly journals Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

2016 ◽  
Vol 473 (23) ◽  
pp. 4473-4491 ◽  
Author(s):  
Ka Wai Fung ◽  
David W. Wright ◽  
Jayesh Gor ◽  
Marcus J. Swann ◽  
Stephen J. Perkins
Keyword(s):  
Crystal Structure ◽  
Domain Structure ◽  
Conformational Changes ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Salt Bridge ◽  
X Ray ◽  
X Ray Scattering ◽  
Nacl Concentration ◽  
Dual Polarisation Interferometry

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg104–Glu1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar RG values of 4.89–4.90 nm for C4b in 137–250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137–250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED–MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b.

scholarly journals Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB–NqrD subunit interface

2021 ◽  
Author(s):  
Alexander Dibrov ◽  
Muntahi Mourin ◽  
Pavel Dibrov ◽  
Grant N. Pierce
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Dynamics Modeling ◽  
Nadh:Quinone Oxidoreductase ◽  
Molecular Dynamics Modeling ◽  
Subunit Interface ◽  
Manual Methods ◽  
The Stability ◽  
Dynamics Simulations

AbstractThe Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB–D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB–D interface underwent a non-physiological transition after 350 ns.

scholarly journals How well do force fields capture the strength of salt bridges in proteins?

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4967 ◽  
Author(s):  
Mustapha Carab Ahmed ◽  
Elena Papaleo ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Molecular Dynamics ◽  
Force Field ◽  
Force Fields ◽  
Salt Bridge ◽  
Dynamics Simulation ◽  
Ionic Interactions ◽  
Salt Bridges ◽  
Important Interaction ◽  
The Stability ◽  
Dynamics Simulations

Salt bridges form between pairs of ionisable residues in close proximity and are important interactions in proteins. While salt bridges are known to be important both for protein stability, recognition and regulation, we still do not have fully accurate predictive models to assess the energetic contributions of salt bridges. Molecular dynamics simulation is one technique that may be used study the complex relationship between structure, solvation and energetics of salt bridges, but the accuracy of such simulations depends on the force field used. We have used NMR data on the B1 domain of protein G (GB1) to benchmark molecular dynamics simulations. Using enhanced sampling simulations, we calculated the free energy of forming a salt bridge for three possible lysine-carboxylate ionic interactions in GB1. The NMR experiments showed that these interactions are either not formed, or only very weakly formed, in solution. In contrast, we show that the stability of the salt bridges is overestimated, to different extents, in simulations of GB1 using seven out of eight commonly used combinations of fixed charge force fields and water models. We also find that the Amber ff15ipq force field gives rise to weaker salt bridges in good agreement with the NMR experiments. We conclude that many force fields appear to overstabilize these ionic interactions, and that further work may be needed to refine our ability to model quantitatively the stability of salt bridges through simulations. We also suggest that comparisons between NMR experiments and simulations will play a crucial role in furthering our understanding of this important interaction.

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