Alpha-Synuclein-Nanoparticle Interactions: Understanding, Controlling and Exploiting Conformational Plasticity

Alpha-synuclein (alpha S) is an extensively studied protein due to its involvement in a group of neurodegenerative disorders, including Parkinson ' s disease, and its documented ability to undergo aberrant self-aggregation resulting in the formation of amyloid-like fibrils. In dilute solution, the protein is intrinsically disordered but can adopt multiple alternative conformations under given conditions, such as upon adsorption to nanoscale surfaces. The study of alpha S-nanoparticle interactions allows us to better understand the behavior of the protein and provides the basis for developing systems capable of mitigating the formation of toxic aggregates as well as for designing hybrid nanomaterials with novel functionalities for applications in various research areas. In this review, we summarize current progress on alpha S-nanoparticle interactions with an emphasis on the conformational plasticity of the biomolecule

Alterations in calmodulin-cardiac ryanodine receptor molecular recognition in congenital arrhythmias

Calmodulin (CaM), a ubiquitous and highly conserved Ca2+-sensor protein involved in the regulation of over 300 molecular targets, has been recently associated with severe forms of lethal arrhythmia. Here, we investigated how arrhythmia-associated mutations in CaM localized at the C-terminal lobe alter the molecular recognition with Ryanodine receptor 2 (RyR2), specifically expressed in cardiomyocytes. We performed an extensive structural, thermodynamic, and kinetic characterization of the variants D95V/H in the EF3 Ca2+-binding motif and of the D129V and D131H/E variants in the EF4 motif, and probed their interaction with RyR2. Our results show that the specific structural changes observed for individual CaM variants do not extend to the complex with the RyR2 target. Indeed, some common alterations emerge at the protein-protein interaction level, suggesting the existence of general features shared by the arrhythmia-associated variants. All mutants showed a faster rate of dissociation from the target peptide than wild-type CaM. Integration of spectroscopic data with exhaustive molecular dynamics simulations suggests that, in the presence of Ca2+, functional recognition involves allosteric interactions initiated by the N-terminal lobe of CaM, which shows a lower affinity for Ca2+ compared to the C-terminal lobe in the isolated protein

Ubistatins Inhibit Proteasome-Dependent Degradation by Binding the Ubiquitin Chain

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys^(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system

Noncanonical sortase-mediated assembly of pilus type 2b in group B Streptococcus

Group B Streptococcus (GBS) expresses 3 structurally distinct pilus types (1, 2a, and 2b) identified as important virulence factors and vaccine targets. These pili are heterotrimeric polymers, covalently assembled on the cell wall by sortase (Srt) enzymes. We investigated the pilus-2b biogenesis mechanism by using a multidisciplinary approach integrating genetic, biochemical, and structural studies to dissect the role of the 2 pilus-2b-associated Srts. We show that only 1 sortase (SrtC1-2b) is responsible for pilus protein polymerization, whereas the second one (Srt2-2b) does not act as a pilin polymerase, but similarly to the housekeeping class A Srt (SrtA), it is involved in cell-wall pilus anchoring by targeting the minor ancillary subunit. Based on its function and sequence features, Srt2-2b does not belong to class C Srts (SrtCs), nor is it a canonical member of any other known family of Srts. We also report the crystal structure of SrtC1-2b at 1.9 Å resolution. The overall fold resembles the typical structure of SrtCs except for the N-terminal lid region that appears in an open conformation displaced from the active site. Our findings reveal that GBS pilus type 2b biogenesis differs significantly from the current model of pilus assembly in gram-positive pathogens.-Lazzarin, M., Cozzi, R., Malito, E., Martinelli, M., D'Onofrio, M., Maione, D., Margarit, I., and Rinaudo, C. D. Noncanonical sortase-mediated assembly of pilus type 2b in group B Streptococcus

Solution NMR insights into dynamic supramolecular assemblies of disordered amyloidogenic proteins

The extraordinary flexibility and structural heterogeneity of intrinsically disordered proteins (IDP) make them functionally versatile molecules. We have now begun to better understand their fundamental role in biology, however many aspects of their behaviour remain difficult to grasp experimentally. This is especially true for the intermolecular interactions which lead to the formation of transient or highly dynamic supramolecular self-assemblies, such as oligomers, aggregation intermediates and biomolecular condensates. Both the emerging functions and pathogenicity of these structures have stimulated great efforts to develop methodologies capable of providing useful insights. Significant progress in solution NMR spectroscopy has made this technique one of the most powerful to describe structural and dynamic features of IDPs within such assemblies at atomic resolution. Here, we review the most recent works that have illuminated key aspects of IDP assemblies and contributed significant advancements towards our understanding of the complex conformational landscape of prototypical disease-associated proteins. We also include a primer on some of the fundamental and innovative NMR methods being used in the discussed studies

Alzheimer's disease-associated ubiquitin mutant Ubb+1: Properties of the carboxy-terminal domain and its influence on biomolecular interactions

Ubb+1, a ubiquitin (Ub) mutant protein originating from misreading of the Ub B gene, is found accumulated in brain tissues of Alzheimer's disease patients. The mutant attracts strong interest due to its possible participation in the molecular events leading to neurodegeneration. Ubb+1 is composed of the globular domain of Ub, linked to a 19-residue C-terminal peptide. Based on NMR relaxation and solvent accessibility measurements we obtained new insight into the molecular properties of Ubb+1. We further determined the thermal stability of Ubb+1 in the monomeric form, and in Lys48- and Lys63-linked dimers. Finally, we explored the influence of the C-terminal fragment on the interactions of Ubb+1 with an isolated UBA2 domain and with membrane mimics. Our data indicate that the C-terminal fragment of Ubb+1 is overall highly flexible, except for a short stretch which appears less solvent-exposed. While influencing the hydrodynamic properties of the globular domain, the fragment does not establish long-lived interactions with the globular domain. It results that the structure and stability of Ub are minimally perturbed by the peptide extension. However, binding to UBA2 and to membrane mimics are both affected, exemplifying possible changes in biomolecular recognition experienced by the disease-associated Ubb+1 compared to the wild-type protein

Preferential Binding of Mg2+ Over Ca2+ to CIB2 Triggers an Allosteric Switch Impaired in Usher Syndrome Type 1J

Calcium and integrin binding protein 2 (CIB2) shares with the other members of the CIB family the ability to bind Ca2+ and Mg2+ via two functional EF-hand motifs, namely EF3 and EF4. As a cation sensor, CIB2 is able to switch to a conformation likely associated with specific biological functions yet to be clarified. Recent findings demonstrate the involvement of CIB2 in hearing physiology and a single, conservative point mutation (p.E64D) has been related to Usher Syndrome type 1J (USH1J) and non-syndromic hearing loss. We present an exhaustive biochemical and biophysical characterization of human wild type (WT) and E64D CIB2. We found that CIB2 does not possibly work as a calcium sensor under physiological conditions, its affinity for Ca2+ (Kdapp = 0.5 mM) being too low for detecting normal intracellular levels. Instead, CIB2 displays a significantly high affinity for Mg2+ (Kdapp = 290 μM), and it is probably Mg2+ -bound under physiological conditions. At odds with the homologous protein CIB1, CIB2 forms a non-covalent dimer under conditions that mimic the physiological ones, and as such it interacts with its physiological target α7B integrin. NMR spectroscopy revealed a long-range allosteric communication between the residue E64, located at the N-terminal domain, and the metal cation binding site EF3, located at the C-terminal domain. The conservative E64D mutation breaks up such inter-domain communication resulting in the impaired ability of CIB2 to switch to its Mg2+-bound form. The ability to bind the target integrin peptide was substantially conserved for E64D CIB2, thus suggesting that the molecular defect associated with USH1J resides in its inability to sense Mg2+ and adopt the required conformation

Dynamics of a Globular Protein Adsorbed to Liposomal Nanoparticles

International audienceA solution-state NMR method is proposed to investigate the dynamics of proteins that undergo reversible association with nanoparticles (NPs). We applied the recently developed dark-state exchange saturation transfer experiment to obtain residue-level dynamic information on a NP-adsorbed protein in the form of transverse spin relaxation rates, R-2(bound). Based on dynamic light scattering, fluorescence, circular dichroism, and NMR spectroscopy data, we show that the test protein, human liver fatty acid binding protein, interacts reversibly and peripherally with liposomal NPs without experiencing significant structural changes. The significant but modest saturation transfer from the bound state observed at 14.1 and 23.5 T static magnetic fields, and the small determined R-2(bound) values were consistent with a largely unrestricted global motion at the lipid surface. Amino acid residues displaying faster spin relaxation mapped to a region that could represent the epitope of interaction with an extended phospholipid chain constituting the protein anchor. These results prove that atomic-resolution protein dynamics is accessible even after association with NPs, supporting the use of saturation transfer methods as powerful tools in bionanoscience

Biophysical and biochemical characterization of Arabidopsis thaliana Calmodulin-like protein CML14

Calcium (Ca2+) is one of the most important second messengers in eukaryotes. Ca2+ binding proteins can be subdivided into two categories: \u201cCa2+ buffers\u201d that modulate Ca2+ ion concentrations in cells, and \u201cCa2+ sensors\u201d that decode Ca2+ signals in a wide array ofphysiological processes in response to external stimuli. Calmodulin (CaM) is the prototypicalexample of Ca2+ sensor proteins in both animals and plants. In addition to conserved CaM,plants possess a unique family of 50 CaM-like proteins (CMLs). Many of these CMLs still remainuncharacterized and the investigation of their biochemical and biophysical properties willprovide insight into Ca2+ signalling in plants. Herein, a detailed characterization of Arabidopsisthaliana CML14 is reported. CML14 is a protein of 148 amino acids with a theoretical molecularweight of 16,579 Da and 50% amino acid sequence identity with AtCaM2. CML14 is predictedto have one functional Ca2+ binding site despite the presence of three EF-hand motifs(Prosite). We overexpressed CML14 in E. coli and analyzed its biochemical and biophysicalcharacteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation,thermal stability and proteolytic susceptibility upon Ca2+ binding. Isothermal titrationcalorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy identified one Ca2+binding site in CML14 and showed that Ca2+ and Mg2+ compete for the same binding site. TheKd values determined by ITC established that CML14 has higher affinity for Ca2+ than forMg2+. Our data were consistent with the sequence based prediction of one functional calciumbinding site. Differential scanning calorimetry (DSC) showed that Ca2+ and Mg2+ have thesame stabilizing effects on protein folding. Apo-CML14 undergoes two thermal unfoldingtransitions, but in the presence of Ca2+ or Mg2+ only one unfolding event at an intermediatetemperature occurs. Limited proteolysis experiments showed that Ca2+ binding affordsprotection against CML14 digestion by trypsin. Surprisingly, CML14 exhibits very fewconformational changes upon calcium binding, which were evaluated by ANS fluorescence andStokes radius measurements in the apo- and Ca2+ bound-forms. These results suggest thatCML14 does not show the characteristics of a classical Ca2+ sensor protein. To betterunderstand the physiological role of CML14 in plants, in vivo analysis will be performed

Solution structure and backbone dynamics of the Cu(I) and apo forms of the second metal-binding domain of the Menkes protein ATP7A.

The second domain of the human Menkes protein (MNK2), formed by 72 residues, has been expressed in Escherichia coli, and its structure has been determined by NMR in both the apo and copper-loaded forms. The structures, obtained with (13)C- and (15)N-labeled samples, are of high quality with backbone rmsd values of 0.51 and 0.41 A and CYANA target functions of 0.39 and 0.38 A(2), respectively. The loop involved in copper binding is part of a hydrophobic patch, which is maintained in both forms. Conformational mobility is observed in the apo form in the same loop. A comparison with metallochaperones and soluble domains of P-type ATPases allows us to relate the primary structure to the occurrence of structural rearrangements upon copper binding