Alessandro Cembran

The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure

Background: The interaction between methionine and aromatic residues in protein complexes is poorly understood. Results: The Met-aromatic motif is prevalent in known protein structures and stabilizes TNF ligand-receptor binding interactions. Conclusion: The Met sulfur-aromatic binding motif provides additional stabilization over purely hydrophobic interactions and at longer distances. Significance: This motif is prevalent and may be associated with a number of mutation- and age-associated diseases. Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.

Structure of the Conical Intersections Driving the cis–trans Photoisomerization of Conjugated Molecules¶

Abstract High-level ab initio calculations show that the singlet photochemical cis–trans isomerization of organic molecules under isolated conditions can occur according to two distinct mechanisms. These mechanisms are characterized by the different structures of the conical intersection funnels controlling photoproduct formation. In nonpolar (e.g. hydrocarbon) polyenes the lowest-lying funnel corresponds to a (CH)3 kink with both double and adjacent single bonds twisted, which may initiate hula-twist (HT) isomerization. On the other hand, in polar conjugated systems such as protonated Schiff bases (PSB) the funnel shows a structure with just one twisted double bond. The ground-state relaxation paths departing from the funnels indicate that the HT motion may take place in nonpolar conjugated systems but also that the single-bond twist may be turned back, whereas in free conjugated polar molecules such as PSB a one-bond flip mechanism dominates from the beginning. The available experimental evidence either supports these predictions or is at least consistent with them.

Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics.

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for ∼70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.

Aborted double bicycle-pedal isomerization with hydrogen bond breaking is the primary event of bacteriorhodopsin proton pumping

Quantum mechanics/molecular mechanics calculations based on ab initio multiconfigurational second order perturbation theory are employed to construct a computer model of Bacteriorhodopsin that reproduces the observed static and transient electronic spectra, the dipole moment changes, and the energy stored in the photocycle intermediate K. The computed reaction coordinate indicates that the isomerization of the retinal chromophore occurs via a complex motion accounting for three distinct regimes: (i) production of the excited state intermediate I, (ii) evolution of I toward a conical intersection between the excited state and the ground state, and (iii) formation of K. We show that, during stage ii, a space-saving mechanism dominated by an asynchronous double bicycle-pedal deformation of the C10═C11─C12═C13─C14═N moiety of the chromophore dominates the isomerization. On this same stage a N─H/water hydrogen bond is weakened and initiates a breaking process that is completed during stage iii.

Connecting Protein Conformational Dynamics with Catalytic Function As Illustrated in Dihydrofolate Reductase

Combined quantum mechanics/molecular mechanics molecular dynamics simulations reveal that the M20 loop conformational dynamics of dihydrofolate reductase (DHFR) is severely restricted at the transition state of the hydride transfer as a result of the M42W/G121V double mutation. Consequently, the double-mutant enzyme has a reduced entropy of activation, i.e., increased entropic barrier, and altered temperature dependence of kinetic isotope effects in comparison with those of wild-type DHFR. Interestingly, in both wild-type DHFR and the double mutant, the average donor-acceptor distances are essentially the same in the Michaelis complex state (~3.5 Å) and the transition state (2.7 Å). It was found that an additional hydrogen bond is formed to stabilize the M20 loop in the closed conformation in the M42W/G121V double mutant. The computational results reflect a similar aim designed to knock out precisely the dynamic flexibility of the M20 loop in a different double mutant, N23PP/S148A.

A refinement protocol to determine structure, topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints

To fully describe the fold space and ultimately the biological function of membrane proteins, it is necessary to determine the specific interactions of the protein with the membrane. This property of membrane proteins that we refer to as structural topology cannot be resolved using X-ray crystallography or solution NMR alone. In this article, we incorporate into XPLOR-NIH a hybrid objective function for membrane protein structure determination that utilizes solution and solid-state NMR restraints, simultaneously defining structure, topology, and depth of insertion. Distance and angular restraints obtained from solution NMR of membrane proteins solubilized in detergent micelles are combined with backbone orientational restraints (chemical shift anisotropy and dipolar couplings) derived from solid-state NMR in aligned lipid bilayers. In addition, a supplementary knowledge-based potential, Ez (insertion depth potential), is used to ensure the correct positioning of secondary structural elements with respect to a virtual membrane. The hybrid objective function is minimized using a simulated annealing protocol implemented into XPLOR-NIH software for general use.

On the Function of Pentameric Phospholamban: Ion Channel or Storage Form?

Phospholamban (PLN) is an integral membrane protein that inhibits the sarcoplasmic reticulum Ca(2+)-ATPase, thereby regulating muscle contractility. We report a combined electrochemical and theoretical study demonstrating that the pentameric PLN does not possess channel activity for conducting chloride or calcium ions across the lipid membrane. This suggests that the pentameric configuration of PLN primarily serves as a storage form for the regulatory function of muscle relaxation by the PLN monomer.

Structure of the intersection space associated with Z/E photoisomerization of retinal in rhodopsin proteins

In this paper we employ a CASSCF/AMBER quantum-mechanics/molecular-mechanics tool to map the intersection space (IS) of a protein. In particular, we provide evidence that the S1 excited-state potential-energy surface of the visual photoreceptor rhodopsin is spanned by an IS segment located right at the bottom of the surface. Analysis of the molecular structures of the protein chromophore (a protonated Schiff base of retinal) along IS reveals a type of geometrical deformation not observed in vacuo. Such a structure suggests that conical intersections mediating different photochemical reactions reside along the same intersection space. This conjecture is investigated by mapping the intersection space of the rhodopsin chromophore model 2-Z-hepta-2,4,6-trieniminium cation and of the conjugated hydrocarbon 3-Z-deca-1,3,5,6,7-pentaene.

Synchronous Opening and Closing Motions Are Essential for cAMP-Dependent Protein Kinase A Signaling

Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and closing of the active cleft without changing the enzymes structure, rendering it catalytically inefficient. Since the opening and closing motions govern the rate-determining product release, we conclude that optimal and coherent conformational fluctuations are necessary for efficient turnover of protein kinases.

Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes

We report molecular dynamics simulations in the explicit membrane environment of a small membrane-embedded protein, sarcolipin, which regulates the sarcoplasmic reticulum Ca-ATPase activity in both cardiac and skeletal muscle. In its monomeric form, we found that sarcolipin adopts a helical conformation, with a computed average tilt angle of 28 +/- 6 degrees and azymuthal angles of 66 +/- 22 degrees, in reasonable accord with angles determined experimentally (23 +/- 2 degrees and 50 +/- 4 degrees, respectively) using solid-state NMR with separated-local-field experiments. The effects of time and spatial averaging on both (15)N chemical shift anisotropy and (1)H/(15)N dipolar couplings have been analyzed using short-time averages of fast amide out-of-plane motions and following principal component dynamic trajectories. We found that it is possible to reproduce the regular oscillatory patterns observed for the anisotropic NMR parameters (i.e., PISA wheels) employing average amide vectors. This work highlights the role of molecular dynamics simulations as a tool for the analysis and interpretation of solid-state NMR data.