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 The structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin

2019 ◽  
Author(s):  
Marte Innselset Flydal ◽  
Martín Alcorlo-Pagés ◽  
Fredrik Gullaksen Johannessen ◽  
Siseth Martínez-Caballero ◽  
Lars Skjærven ◽  
...  
Keyword(s):  
Active Site ◽  
Phenylalanine Hydroxylase ◽  
Genetic Disorder ◽  
3D Structure ◽  
Interaction Network ◽  
Salt Bridge ◽  
Data Bank ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Pharmacological Chaperone

AbstractPhenylalanine 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 the first structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the pre-catalytic state. Crystal structures, solved at 3.18Å 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 pre-catalytic towards the active conformation, a molecular mechanism that was supported by site directed mutagenesis and targeted MD 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.Significance StatementThe present crystal structure of phenylalanine hydroxylase (PAH) provides the first and long-awaited 3D-structure of the full-length human PAH, both unbound and complexed with the tetrahydrobiopterin (BH4) cofactor.The BH4-bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH4 (Kuvan®) is the only drug-based therapy for a subset of phenylketonuria patients.We found two tetramer conformations in the same crystal, depending on the active site occupation by BH4, which aid to understand the stabilization by BH4 and the allosteric mechanisms in PAH. The structure also reveals the increased mobility of human-compared with rat PAH, in line with an increased predisposition to disease-associated mutations in human.Classification: Biological Science (major), biochemistry (minor)The authors declare no conflict of interest.Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6HYC and 6HPO). The EM maps have been deposited in the EMDB with accession s EMD-4605This article contains supporting information.

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 Modulating long-range energetics via helix stabilization: a case study using T4 lysozyme

2018 ◽  
Author(s):  
Sabriya N. Rosemond ◽  
Kambiz M. Hamadani ◽  
Jamie H.D. Cate ◽  
Susan Marqusee
Keyword(s):  
Protein Folding ◽  
Long Range ◽  
Salt Bridge ◽  
Globular Protein ◽  
Full Length ◽  
T4 Lysozyme ◽  
Long Distance ◽  
Terminal Fragment ◽  
The Stability

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization.

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.

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.

The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX)4 and Coumarin-RXDX Conjugate β-Sheets

2020 ◽  
Author(s):  
Ryan Weber ◽  
Martin McCullagh
Keyword(s):  
Biological Imaging ◽  
Ph Sensing ◽  
Hydrophobic Residue ◽  
Ideal Candidate ◽  
Self Assembling ◽  
Sensing Applications ◽  
Β Sheets ◽  
The Stability ◽  
Dynamics Simulations

<p>pH-switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X=Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX)<sub>4</sub> systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X=Ala are found to be unstable and both pHs while conjugates with X=Val, Leu, Ile and Phe are found to form stable β-sheets at least at neutral pH. The (RFDF)<sub>4</sub>-coumarin conjugate is found to have the largest relative entropy value of 0.884 +/- 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF)<sub>4</sub> containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.</p>

Ion Pairing and Dielectric Decrement in Glycosaminoglycan Brushes

2020 ◽  
Author(s):  
James Sterling ◽  
Wenjuan Jiang ◽  
Wesley M. Botello-Smith ◽  
Yun L. Luo
Keyword(s):  
Molecular Dynamics ◽  
Ion Pairs ◽  
Mean Field ◽  
Salt Bridge ◽  
Ion Pairing ◽  
Donnan Potential ◽  
Mean Field Models ◽  
Ion Exclusion ◽  
Dynamics Simulations ◽  
Contact Ion Pairs

Molecular dynamics simulations of hyaluronic acid and heparin brushes are presented that show important effects of ion-pairing, water dielectric decrease, and co-ion exclusion. Results show equilibria with electroneutrality attained through screening and pairing of brush anionic charges by cations. Most surprising is the reversal of the Donnan potential that would be expected based on electrostatic Boltzmann partitioning alone. Water dielectric decrement within the brush domain is also associated with Born hydration-driven cation exclusion from the brush. We observe that the primary partition energy attracting cations to attain brush electroneutrality is the ion-pairing or salt-bridge energy associated with cation-sulfate and cation-carboxylate solvent-separated and contact ion pairs. Potassium and sodium pairing to glycosaminoglycan carboxylates and sulfates consistently show similar abundance of contact-pairing and solvent-separated pairing. In these crowded macromolecular brushes, ion-pairing, Born-hydration, and electrostatic potential energies all contribute to attain electroneutrality and should therefore contribute in mean-field models to accurately represent brush electrostatics.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

Applications of random graphs

2017 ◽  
Author(s):  
A.C.C. Coolen ◽  
A. Annibale ◽  
E.S. Roberts
Keyword(s):  
Social Networks ◽  
Random Graphs ◽  
Interaction Network ◽  
Power Grids ◽  
Protein Protein Interaction ◽  
Topological Features ◽  
First Case ◽  
The Stability ◽  
Protein Protein Interaction Network

This chapter reviews graph generation techniques in the context of applications. The first case study is power grids, where proposed strategies to prevent blackouts have been tested on tailored random graphs. The second case study is in social networks. Applications of random graphs to social networks are extremely wide ranging – the particular aspect looked at here is modelling the spread of disease on a social network – and how a particular construction based on projecting from a bipartite graph successfully captures some of the clustering observed in real social networks. The third case study is on null models of food webs, discussing the specific constraints relevant to this application, and the topological features which may contribute to the stability of an ecosystem. The final case study is taken from molecular biology, discussing the importance of unbiased graph sampling when considering if motifs are over-represented in a protein–protein interaction network.

scholarly journals Rational thermostabilisation of four-helix bundle dimeric de novo proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shin Irumagawa ◽  
Kaito Kobayashi ◽  
Yutaka Saito ◽  
Takeshi Miyata ◽  
Mitsuo Umetsu ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Protein Complexes ◽  
Salt Bridge ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Helix Bundle ◽  
Stable Mutant ◽  
Four Helix Bundle ◽  
The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

Sign in / Sign up

Export Citation Format

Share Document