Johnny Habchi

Introducing Protein Intrinsic Disorder

Johnny Habchi,†,‡ Peter Tompa,* Sonia Longhi,†,‡,* and Vladimir N. Uversky* †Aix-Marseille Universite,́ Architecture et Fonction des Macromolećules Biologiques (AFMB), UMR 7257, 13288, Marseille, France ‡CNRS, Architecture et Fonction des Macromolećules Biologiques (AFMB), UMR 7257, 13288, Marseille, France VIB Department of Structural Biology, Vrije Universiteit Brussel, 1050 Ixelles, Belgium Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, H-1113, Hungary Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33620, United States Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

Structural disorder in viral proteins.

Bin Xue,† David Blocquel, Johnny Habchi, Alexey V. Uversky, Lukasz Kurgan, Vladimir N. Uversky,*,‡,∇ and Sonia Longhi* †Department of Cell Biology, Microbiology and Molecular Biology, College of Fine Arts and Sciences, and ‡Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, Florida 33620, United States Architecture et Fonction des Macromolecules Biologiques (AFMB) UMR 7257, Aix-Marseille Universite, 13288 Marseille, France AFMB UMR 7257, CNRS, 13288 Marseille, France Center for Data Analytics and Biomedical Informatics, Department of Computer and Information Sciences, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, United States Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, Canada T6G 2R3 Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Russia

An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer's disease.

An approved anticancer drug selectively targets the first step in the molecular cascade resulting in Alzheimer’s disease. The conversion of the β-amyloid (Aβ) peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer’s disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Aβ or inhibit its aggregation, all clinical trials with these objectives have so far failed, at least in part because of a lack of understanding of the molecular mechanisms underlying the process of aggregation and its inhibition. To address this problem, we describe a chemical kinetics approach for rational drug discovery, in which the effects of small molecules on the rates of specific microscopic steps in the self-assembly of Aβ42, the most aggregation-prone variant of Aβ, are analyzed quantitatively. By applying this approach, we report that bexarotene, an anticancer drug approved by the U.S. Food and Drug Administration, selectively targets the primary nucleation step in Aβ42 aggregation, delays the formation of toxic species in neuroblastoma cells, and completely suppresses Aβ42 deposition and its consequences in a Caenorhabditis elegans model of Aβ42-mediated toxicity. These results suggest that the prevention of the primary nucleation of Aβ42 by compounds such as bexarotene could potentially reduce the risk of onset of Alzheimer’s disease and, more generally, that our strategy provides a general framework for the rational identification of a range of candidate drugs directed against neurodegenerative disorders.

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (NTAIL) are both intrinsically disordered. Here we show that Henipavirus NTAIL domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (PXD) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that NTAIL and PXD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 m and has a KD in the μm range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that PXD triggers an increase in the α-helical content of NTAIL. Using fluorescence spectroscopy, we show that PXD has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus NTAIL domain, thus arguing for the lack of stable contacts between the C termini of NTAIL and PXD. Finally, we present a tentative structural model of the NTAIL-PXD interaction in which a short, order-prone region of NTAIL (α-MoRE; amino acids 473–493) adopts an α-helical conformation and is embedded between helices α2 and α3 of PXD, leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the NTAIL-PXD interaction as a valuable target for rational antiviral approaches.

Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus.

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, NTAIL, of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered NTAIL domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between NTAIL and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of NTAIL upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to NTAIL without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer’s disease

Significance The absence of fully reproducible protein aggregation assays has contributed to the systematic failures in clinical trials for Alzheimer’s disease (AD) of compounds targeting the aggregation process of the amyloid-β peptide (Aβ). To address this problem, we report the identification of a library of compounds against Aβ aggregation using a drug discovery strategy based on highly quantitative aggregation rate measurements. We then demonstrate, both in Caenorhabditis elegans and human cerebrospinal fluid, that this approach can systematically provide a rich variety of related small molecules to take forward into a drug discovery process. We therefore report an approach that should substantially help overcome the very high level of attrition associated with drug discovery programs for AD. The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer’s disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy

This work aims at characterizing structural transitions within the intrinsically disordered C-terminal domain of the nucleoprotein (NTAIL) from the Nipah and Hendra viruses, two recently emerged pathogens gathered within the Henipavirus genus. To this end, we used site-directed spin labeling combined with electron paramagnetic resonance spectroscopy to investigate the α-helical-induced folding that Henipavirus NTAIL domains undergo in the presence of the C-terminal X domain of the phosphoprotein (PXD). For each NTAIL protein, six positions located within four previously proposed molecular recognition elements (MoREs) were targeted for spin labeling, with three of these positions (475, 481, and 487) falling within the MoRE responsible for binding to PXD (Box3). A detailed analysis of the impact of the partner protein on the labeled NTAIL variants revealed a dramatic modification in the environment of the spin labels grafted within Box3, with the observed modifications supporting the formation of an induced α-helix within this region. In the free state, the slightly lower mobility of the spin labels grafted within Box3 as compared to the other positions suggests the existence of a transiently populated α-helix, as already reported for measles virus (MeV) NTAIL. Comparison with the well-characterized MeV NTAIL–PXD system, allowed us to validate the structural models of Henipavirus NTAIL–PXD complexes that we previously proposed. In addition, this study highlighted a few notable differences between the Nipah and Hendra viruses. In particular, the observation of composite spectra for the free form of the Nipah virus NTAIL variants spin labeled in Box3 supports conformational heterogeneity of this partly pre-configured α-helix, with the pre-existence of stable α-helical segments. Altogether these results provide insights into the molecular mechanisms of the Henipavirus NTAIL–PXD binding reaction.

The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments

The free energy landscape theory has been very successful in rationalizing the folding behaviour of globular proteins, as this representation provides intuitive information on the number of states involved in the folding process, their populations and pathways of interconversion. We extend here this formalism to the case of the Aβ40 peptide, a 40-residue intrinsically disordered protein fragment associated with Alzheimer’s disease. By using an advanced sampling technique that enables free energy calculations to reach convergence also in the case of highly disordered states of proteins, we provide a precise structural characterization of the free energy landscape of this peptide. We find that such landscape has inverted features with respect to those typical of folded proteins. While the global free energy minimum consists of highly disordered structures, higher free energy regions correspond to a large variety of transiently structured conformations with secondary structure elements arranged in several different manners, and are not separated from each other by sizeable free energy barriers. From this peculiar structure of the free energy landscape we predict that this peptide should become more structured and not only more compact, with increasing temperatures, and we show that this is the case through a series of biophysical measurements.

Molecular basis for structural heterogeneity of an intrinsically disordered protein bound to a partner by combined ESI-IM-MS and modeling

AbstractIntrinsically disordered proteins (IDPs) form biologically active complexes that can retain a high degree of conformational disorder, escaping structural characterization by conventional approaches. An example is offered by the complex between the intrinsically disordered NTAIL domain and the phosphoprotein X domain (PXD) from measles virus (MeV). Here, distinct conformers of the complex are detected by electrospray ionization-mass spectrometry (ESI-MS) and ion mobility (IM) techniques yielding estimates for the solvent-accessible surface area (SASA) in solution and the average collision cross-section (CCS) in the gas phase. Computational modeling of the complex in solution, based on experimental constraints, provides atomic-resolution structural models featuring different levels of compactness. The resulting models indicate high structural heterogeneity. The intermolecular interactions are predominantly hydrophobic, not only in the ordered core of the complex, but also in the dynamic, disordered regions. Electrostatic interactions become involved in the more compact states. This system represents an illustrative example of a hydrophobic complex that could be directly detected in the gas phase by native mass spectrometry. This work represents the first attempt to modeling the entire NTAIL domain bound to PXD at atomic resolution. Graphical Abstractᅟ

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus

Background: Binding of the MeV C-terminal disordered domain of the nucleoprotein (NTAIL) to the X domain (XD) of the phosphoprotein mediates recruitment of the polymerase. Results: NTAIL amino acid substitutions that reduce NTAIL-XD affinity and/or NTAIL α-helical folding do not affect polymerase rates but strongly affect infectivity. Conclusion: MeV polymerase tolerates NTAIL amino acid substitutions. Significance: NTAIL sequence plays a role in optimal infectivity. The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. Binding of P to nucleocapsid is mediated by the X domain of P (XD) and a conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (NTAIL). XD binding induces NTAIL α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assessed the relationships among XD-induced NTAIL folding, XD-NTAIL binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced NTAIL α-helical folding were created within Box-2 of Edmonston MeV NTAIL. Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-NTAIL binding affinity or reduction/loss of XD-induced NTAIL alpha-helical folding. Recombinant infectious virus was recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent virus. Box-2 mutations did however impose a significant cost to infectivity, reflected in an increase in the amount of input genome required to match the infectivity of parent virus. Diminished infectivity could not be attributed to changes in virion protein composition or production of defective interfering particles, where changes from parent virus were within a 3-fold range. The results indicated that MeV polymerase activity, but not infectivity, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.