Visualizing protein breathing motions associated with aromatic ring flipping.

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold1-3. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.

NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B.

Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.

A specific phosphorylation-dependent conformational switch in SARS-CoV-2 nucleocapsid protein inhibits RNA binding.

The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.

The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a.

The processes of genome replication and transcription of SARS-CoV-2 represent important targets for viral inhibition. Betacoronaviral nucleoprotein (N) is a highly dynamic cofactor of the replication-transcription complex (RTC), whose function depends on an essential interaction with the amino-terminal ubiquitin-like domain of nsp3 (Ubl1). Here, we describe this complex (dissociation constant - 30 to 200 nM) at atomic resolution. The interaction implicates two linear motifs in the intrinsically disordered linker domain (N3), a hydrophobic helix (219LALLLLDRLNQL230) and a disordered polar strand (243GQTVTKKSAAEAS255), that mutually engage to form a bipartite interaction, folding N3 around Ubl1. This results in substantial collapse in the dimensions of dimeric N, forming a highly compact molecular chaperone, that regulates binding to RNA, suggesting a key role of nsp3 in the association of N to the RTC. The identification of distinct linear motifs that mediate an important interaction between essential viral factors provides future targets for development of innovative strategies against COVID-19.

1H, 13C and 15N Backbone chemical shift assignments of the n-terminal and central intrinsically disordered domains of SARS-CoV-2 nucleoprotein.

The nucleoprotein (N) from SARS-CoV-2 is an essential cofactor of the viral replication transcription complex and as such represents an important target for viral inhibition. It has also been shown to colocalize to the transcriptase-replicase complex, where many copies of N decorate the viral genome, thereby protecting it from the host immune system. N has also been shown to phase separate upon interaction with viral RNA. N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45-175 and 264-365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1-44 and N3, spanning residues 176-263). Our assignment provides the basis for the identification of inhibitors and functional and interaction studies of this essential protein.

The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases.

BACKGROUND: Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. RESULTS: We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. CONCLUSIONS: The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

1H, 13C and 15N backbone chemical shift assignments of SARS-CoV-2 nsp3a.

The non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein.

NMR and circular dichroism data for domain 2 of the HCV NS5A protein phosphorylated by the Casein Kinase II.

The Hepatitis C Virus (HCV) nonstructural 5A protein (NS5A) is a phosphoprotein (Evans et al., 2004; Ross-Thriepland and Harris, 2014) [1], [2] composed of an N-terminal well-structured domain and two C-terminal intrinsically disordered domains (Moradpour et al., 2007; Bartenschlager et al., 2013; Badillo et al., 2017) [3], [4], [5]. So far, no precise molecular function has been identified for this viral protein (Ross-Thriepland and Harris, 2015) [6] which is required for viral replication (Tellinghuisen et al., 2008) [7]. In this article, we present datasets of NMR and circular dichroism analyses of the domain 2 of the HCV NS5A protein (NS5A-D2) phosphorylated in vitro by the Casein Kinase II (CKII) (Dal Pero et al., 2007; Clemens et al., 2015; Masak et al., 2014; Kim et al., 2014) [8], [9], [10], [11]. We describe the in vitro phosphorylation of the serine 288 (pS288) of NS5A-D2 by CKII and report the circular dichroism spectrum of the phosphorylated domain (NS5-D2_CKII). This data article also contains the 1H, 15N and 13C NMR chemical shift assignments (HN, N, Cα, Cß and C') for the phosphorylated NS5A-D2 domain, and an assigned 1H,15N-HSQC spectrum is shown. The NMR data have been acquired on an 800 MHz spectrometer. These NMR data have been used to calculate both the 1H,15N combined chemical shift perturbations (CSP) induced by the phosphorylation of pS288 and the secondary structural propensity (SSP) scores that describe the structural tendencies in this intrinsically disordered domain. The circular dichroism spectrum and the SSP scores of NS5A-D2_CKII have been compared with those of unphosphorylated NS5A-D2 [12,13].

Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment.

In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the "fly-casting" hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

In vitro and in vivo activity of a new unsymmetrical dinuclear copper complex containing a derivative ligand of 1,4,7-triazacyclononane: catalytic promiscuity of [Cu2(L)Cl3].

Here we present the synthesis of the dinuclear complex [Cu(II)2(L)Cl3] (1), where L is the deprotonated form of the 3-[(4,7-diisopropyl-1,4,7-triazacyclononan-1-yl)methyl]-2-hydroxy-5-methylbenzaldehyde ligand. The complex was characterized by single crystal X-ray diffraction, potentiometric titration, mass spectrometry, electrochemical and magnetic measurements, EPR, UV-Vis and IR. Complex 1 is able to increase the hydrolysis rate of the diester bis-(2,4-dinitrophenyl)phosphate (2,4-BDNPP) by a factor of 2700, and also to promote the plasmidial DNA cleavage at pH 6 and to inhibit the formazan chromophore formation in redox processes at pH 7. Using Saccharomyces cerevisiae (BY4741) as a eukaryotic cellular model, we observed that 1 presents reduced cytotoxicity. In addition, treatment of wild-type and mutant cells lacking Cu/Zn-superoxide dismutase (Sod1) and cytoplasmic catalase (Ctt1) with 1 promotes increased survival after H2O2 or menadione (O2˙(-) generator) stress, indicating that 1 might act as a Sod1 and Ctt1 mimetic. Considered together, these results support considerations regarding the dynamic behaviour of an unsymmetrical dinuclear copper(II) complex in solid state and in aqueous pH-dependent solution.