The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.

HACANCOi: a new Hα-detected experiment for backbone resonance assignment of intrinsically disordered proteins.

Unidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T2) relaxation times allowing sophisticated coherence transfer pathway selection in comparison to folded proteins. 1Hα-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs. However, many IDPs undergo a disorder-to-order transition upon interaction with their target protein, which leads to the loss of the favorable relaxation properties. Long coherence transfer routes now result in prohibitively large decrease in sensitivity. We introduce a novel 4D 1Hα-detected experiment HACANCOi, together with its 3D implementation, which warrant high sensitivity for the assignment of proline-rich regions in IDPs in complex with a globular protein. The experiment correlates 1Hαi, 13Cαi, 15Ni and [Formula: see text] spins by transferring the magnetization concomitantly from 13Cαi to 15Ni and [Formula: see text]. The B1 domain of protein G (GB1), and the enteropathogenic E. coli EspF in complex with human SNX9 SH3, serve as model systems to demonstrate the attainable sensitivity and successful sequential assignment.

Dispersion from Cα or NH: 4D experiments for backbone resonance assignment of intrinsically disordered proteins.

Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three Hα-detected 4D experiments with Cα as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D Hα-start, HN-detect experiments which have two NH dimensions to enhance peak dispersion in a sequential walk through C', NH and HN, and provide more accurate NH/HN chemical shifts than those that can be obtained from a crowded 1H, 15N-HSQC spectrum. Application of these 4D experiments is demonstrated using BilRI (165 aa), an outer-membrane intrinsically disordered protein from the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans. BilRI amino acid sequence encompasses three very similar repeats with a 13-residue identical stretch in two of them.

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia.

Mitral valve diseases affect ∼3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from nonsyndromic mitral valve dysplasia (MVD). The filamin A (FLNA) protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD-causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function has mostly remained elusive. In this study, using NMR spectroscopy and interaction assays, we investigated FLNA-MVD-causing V711D and H743P mutations. Our results clearly indicated that both mutations almost completely destroyed the folding of the FLNA5 domain, where the mutation is located, and also affect the folding of the neighboring FLNA4 domain. The structure of the neighboring FLNA6 domain was not affected by the mutations. These mutations also completely abolish FLNA's interactions with protein tyrosine phosphatase nonreceptor type 12, which has been suggested to contribute to the pathogenesis of FLNA-MVD. Taken together, our results provide an essential structural and molecular framework for understanding the molecular bases of FLNA-MVD, which is crucial for the development of new therapies to replace surgery.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.

Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.

Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH.

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ H(N), H(α) or (13)C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with (1)H(N)(i - 1), (13)C'(i - 1) and (15)N(i), and (1)H(N)(i + 1), (13)C'(i) and (15)N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.

Recognition of tandem PxxP motifs as a unique Src homology 3-binding mode triggers pathogen-driven actin assembly.

Src homology 3 (SH3) domains are globular protein interaction modules that regulate cell behavior. The classic SH3 ligand-binding site accommodates a hydrophobic PxxP motif and a positively charged specificity-determining residue. We have determined the NMR structure of insulin receptor tyrosine kinase substrate (IRTKS) SH3 domain in complex with a repeat from Escherichia coli-secreted protein F-like protein encoded on prophage U (EspF(U)), a translocated effector of enterohemorrhagic E. coli that commandeers the mammalian actin assembly machinery. EspF(U)-IRTKS interaction is among the highest affinity natural SH3 ligands. Our complex structure reveals a unique type of SH3 interaction based on recognition of tandem PxxP motifs in the ligand. Strikingly, the specificity pocket of IRTKS SH3 has evolved to accommodate a polyproline type II helical peptide analogously to docking of the canonical PxxP by the conserved IRTKS SH3 proline-binding pockets. This cooperative binding explains the high-affinity SH3 interaction and is required for EspF(U)-IRTKS interaction in mammalian cells as well as the formation of localized actin "pedestals" beneath bound bacteria. Importantly, tandem PxxP motifs are also found in mammalian ligands and have been shown to contribute to IRTKS SH3 recognition similarly.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects neurons and repairs the Parkinson disease-like symptoms in a rat 6-hydroxydopamine model. We show a three-dimensional solution structure of human MANF that differs drastically from other neurotrophic factors. Remarkably, the C-terminal domain of MANF (C-MANF) is homologous to the SAP domain of Ku70, a well known inhibitor of proapoptotic Bax (Bcl-2-associated X protein). Cellular studies confirm that MANF and C-MANF protect neurons intracellularly as efficiently as Ku70.

Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.

Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins.

Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented by utilizing (15)N, (13)C' and (1)H(N) spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues. Especially (15)N and (13)C' spins offer superior chemical shift dispersion in comparison to (13)C(α) and (13)C(ß) spins. However, HN-detected experiments suffer from exchange broadening of amide proton signals on IDPs especially under alkali conditions. To that end, we propose here two novel HA-detected experiments, (HCA)CON(CA)H and (HCA)NCO(CA)H and a new assignment protocol based on panoply of unidirectional HA-detected experiments that enable robust backbone assignment of IDPs also at high pH. The new approach was tested at pH 6.5 and pH 8.5 on cancer/testis antigen CT16, a 110-residue IDP, and virtually complete backbone assignment of CT16 was obtained by employing the novel HA-detected experiments together with the previously introduced iH(CA)NCO scheme. Remarkably, also those 10 N-terminal residues that remained unassigned in our earlier HN-detection based assignment approach even at pH 6.5 were now readily assigned. Moreover, theoretical calculations and experimental results suggest that overall sensitivity of the new experiments is also applicable to small or medium sized globular proteins that require alkaline conditions.

Enteroviruses and coronaviruses: similarities and therapeutic targets.

Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes.Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral proteases have been evaluated.Expert opinion: The main protease of coronaviruses and enteroviruses share similarities in their structure and function. These proteases process their viral polyproteins and thus drugs that bind to the active site have potential to target both virus groups. It is important to develop drugs that target more evolutionarily conserved processes and proteins. Moreover, it is a wise strategy to concentrate on processes that are similar between several virus families.

1H, 13C, and 15N NMR chemical shift assignment of the complex formed by the first EPEC EspF repeat and N-WASP GTPase binding domain.

LEE-encoded effector EspF (EspF) is an effector protein part of enteropathogenic Escherichia coli's (EPEC's) arsenal for intestinal infection. This intrinsically disordered protein contains three highly conserved repeats which together compose over half of the protein's complete amino acid sequence. EPEC uses EspF to hijack host proteins in order to promote infection. In the attack EspF is translocated, together with other effector proteins, to host cell via type III secretion system. Inside host EspF stimulates actin polymerization by interacting with Neural Wiskott-Aldrich syndrome protein (N-WASP), a regulator in actin polymerization machinery. It is presumed that EspF acts by disrupting the autoinhibitory state of N-WASP GTPase binding domain. In this NMR spectroscopy study, we report the 1H, 13C, and 15N resonance assignments for the complex formed by the first 47-residue repeat of EspF and N-WASP GTPase binding domain. These near-complete resonance assignments provide the basis for further studies which aim to characterize structure, interactions, and dynamics between these two proteins in solution.

HA-detected experiments for the backbone assignment of intrinsically disordered proteins.

We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual (15)N(i)-(13)C'(i)-H(alpha)(i) (or (15)N(i)-(13)C(alpha)(i)-H(alpha)(i)) and sequential (15)N(i + 1)-(13)C'(i)-H(alpha)(i) (or (15)N(i + 1)-(13)C(alpha)(i)-H(alpha)(i)) correlation experiments together with efficient use of chemical shifts of (15)N and (13)C' nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspF(U). Using the proposed approach, we were able to assign 90% of (1)H(alpha), (13)C(alpha), (13)C', (15)N chemical shifts in EspF(U). We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.

An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins.

An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely (13)C'(i), (15)N(i), (1)HN(i) connectivities in uniformly 15N/13C-labeled proteins. In comparison to the "out-and-back" style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the (13)C' dimension and on average 1.6 times higher sensitivity especially for residues in alpha-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16.

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

Maternal Inheritance of a Recessive RBP4 Defect in Canine Congenital Eye Disease.

Maternally skewed transmission of traits has been associated with genomic imprinting and oocyte-derived mRNA. We report canine congenital eye malformations, caused by an amino acid deletion (K12del) near the N terminus of retinol-binding protein (RBP4). The disease is only expressed when both dam and offspring are deletion homozygotes. RBP carries vitamin A (retinol) from hepatic stores to peripheral tissues, including the placenta and developing eye, where it is required to synthesize retinoic acid. Gestational vitamin A deficiency is a known risk factor for ocular birth defects. The K12del mutation disrupts RBP folding in vivo, decreasing its secretion from hepatocytes to serum. The maternal penetrance effect arises from an impairment in the sequential transfer of retinol across the placenta, via RBP encoded by maternal and fetal genomes. Our results demonstrate a mode of recessive maternal inheritance, with a physiological basis, and they extend previous observations on dominant-negative RBP4 alleles in humans.

1H, 13C and 15N NMR chemical shift assignments of A. thaliana RCD1 RST.

The A. thaliana RCD1 (radical-induced cell death1) protein is a cellular signaling hub protein which interacts with numerous plant transcription factors from different families. It consists of three conserved domains and intervening unstructured regions, the C-terminal RST domain being responsible for the interactions with the transcription factors. It has been shown that many partner proteins interact with RCD1 RST via their intrinsically disordered regions, and that the domain is able to house partners with divergent folds. We aim to structurally characterize the RCD1 RST domain and its complexes [complex with DREB2A]. Here we report the 1H, 15N and 13C chemical shift assignments of the backbone and sidechain atoms for RCD1 (468-589) containing the RST (510-567) domain.

Skeletal Dysplasia Mutations Effect on Human Filamins' Structure and Mechanosensing.

Cells' ability to sense mechanical cues in their environment is crucial for fundamental cellular processes, leading defects in mechanosensing to be linked to many diseases. The actin cross-linking protein Filamin has an important role in the conversion of mechanical forces into biochemical signals. Here, we reveal how mutations in Filamin genes known to cause Larsen syndrome and Frontometaphyseal dysplasia can affect the structure and therefore function of Filamin domains 16 and 17. Employing X-ray crystallography, the structure of these domains was first solved for the human Filamin B. The interaction seen between domains 16 and 17 is broken by shear force as revealed by steered molecular dynamics simulations. The effects of skeletal dysplasia associated mutations of the structure and mechanosensing properties of Filamin were studied by combining various experimental and theoretical techniques. The results showed that Larsen syndrome associated mutations destabilize or even unfold domain 17. Interestingly, those Filamin functions that are mediated via domain 17 interactions with other proteins are not necessarily affected as strongly interacting peptide binding to mutated domain 17 induces at least partial domain folding. Mutation associated to Frontometaphyseal dysplasia, in turn, transforms 16-17 fragment from compact to an elongated form destroying the force-regulated domain pair.

Effect of protein structure on laccase-catalyzed protein oligomerization.

Laccase-catalyzed oligomerization of proteins was studied using Trametes hirsuta laccase (ThL) and coactosin as a model system. The reaction mechanism was elucidated using free amino acids and the tripeptide Gly-Leu-Tyr as substrates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) as well as oxygen consumption measurements and SDS-PAGE were used to study the reactions. Of the 15 selected amino acids, ThL was found to oxidize tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys), of which the reactions with Tyr and Cys have been described earlier. ThL was able to link four full-length coactosins, whereas coactosin that was truncated from its C-terminus remained unpolymerized. Of the four tyrosine residues present in coactosin, only the tyrosine in the C-terminus was found to be reactive. Polymerization between tyrosine side-chains was unambiguously shown using different oligomers of Gly-Leu-Tyr as parent ions in MALDI-TOF/TOF MS fragment ion analyses.