Solution NMR studies of membrane-protein-chaperone complexes

The biosynthesis of the bacterial outer membrane depends on molecular chaperones that protect hydrophobic membrane proteins against aggregation while transporting them across the periplasm. In our ongoing research, we use high-resolution NMR spectroscopy in aqueous solution as the main technique to characterize the structures and biological functions of these membrane-protein-chaperone complexes. Here, we describe NMR studies addressing three functional aspects of periplasmic membrane-protein-chaperone complexes. Firstly, the Escherichia coli outer membrane protein OmpX binds to each of the two chaperones, Skp and SurA, in structurally at least partially similar states despite fundamental differences between the three-dimensional structures of the chaperones. Secondly, we show that the Skp-bound state of OmpX is equivalent to a chemically denatured state in terms of its refolding competence into detergent micelles in vitro. Thirdly, we use amino acid mutation analysis to show that the interaction of OmpX to Skp is not dominated by the two most hydrophobic segments of OmpX

Chaperones and chaperone-substrate complexes: dynamic playgrounds for NMR spectroscopists

The majority of proteins depend on a well-defined three-dimensional structure to obtain their functionality. In the cellular environment, the process of protein folding is guided by molecular chaperones to avoid misfolding, aggregation, and the generation of toxic species. To this end, living cells contain complex networks of molecular chaperones, which interact with substrate polypeptides by a multitude of different functionalities: transport them towards a target location, help them fold, unfold misfolded species, resolve aggregates, or deliver them towards a proteolysis machinery. Despite the availability of high-resolution crystal structures of many important chaperones in their substrate-free apo forms, structural information about how substrates are bound by chaperones and how they are protected from misfolding and aggregation is very sparse. This lack of information arises from the highly dynamic nature of chaperone-substrate complexes, which so far has largely hindered their crystallization. This highly dynamic nature makes chaperone-substrate complexes good targets for NMR spectroscopy. Here, we review the results achieved by NMR spectroscopy to understand chaperone function in general and details of chaperone-substrate interactions in particular. We assess the information content and applicability of different NMR techniques for the characterization of chaperones and chaperone-substrate complexes. Finally, we highlight three recent studies, which have provided structural descriptions of chaperone-substrate complexes at atomic resolution

Bispecific Tau Antibodies with Additional Binding to C1q or Alpha-Synuclein

BACKGROUND: Alzheimer’s disease (AD) and other tauopathies are neurodegenerative disorders characterized by cellular accumulation of aggregated tau protein. Tau pathology within these disorders is accompanied by chronic neuroinflammation, such as activation of the classical complement pathway by complement initiation factor C1q. Additionally, about half of the AD cases present with inclusions composed of aggregated alpha-synuclein called Lewy bodies. Lewy bodies in disorders such as Parkinson’s disease and Lewy body dementia also frequently occur together with tau pathology. OBJECTIVE: Immunotherapy is currently the most promising treatment strategy for tauopathies. However, the presence of multiple pathological processes within tauopathies makes it desirable to simultaneously target more than one disease pathway. METHODS: Herein, we have developed three bispecific antibodies based on published antibody binding region sequences. One bispecific antibody binds to tau plus alpha-synuclein and two bispecific antibodies bind to tau plus C1q. RESULTS: Affinity of the bispecific antibodies to their targets compared to their monospecific counterparts ranged from nearly identical to one order of magnitude lower. All bispecific antibodies retained binding to aggregated protein in patient-derived brain sections. The bispecific antibodies also retained their ability to inhibit aggregation of recombinant tau, regardless of whether the tau binding sites were in IgG or scFv format. Mono- and bispecific antibodies inhibited cellular seeding induced by AD-derived pathological tau with similar efficacy. Finally, both Tau-C1q bispecific antibodies completely inhibited the classical complement pathway. CONCLUSIONS: Bispecific antibodies that bind to multiple pathological targets may therefore present a promising approach to treat tauopathies and other neurodegenerative disorders

Structural determinants of multimerization and dissociation in 2-Cys peroxiredoxin chaperone function

Peroxiredoxins (PRDXs) are abundant peroxidases present in all kingdoms of life. Recently, they have been shown to also carry out additional roles as molecular chaperones. To address this emerging supplementary function, this review focuses on structural studies of 2-Cys PRDX systems exhibiting chaperone activity. We provide a detailed understanding of the current knowledge of structural determinants underlying the chaperone function of PRDXs. Specifically, we describe the mechanisms which may modulate their quaternary structure to facilitate interactions with client proteins and how they are coordinated with the functions of other molecular chaperones. Following an overview of PRDX molecular architecture, we outline structural details of the presently best-characterized peroxiredoxins exhibiting chaperone function and highlight common denominators. Finally, we discuss the remarkable structural similarities between 2-Cys PRDXs, small HSPs, and J-domain-independent Hsp40 holdases in terms of their functions and dynamic equilibria between low- and high-molecular-weight oligomers

Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification

Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (αinh) that partly obstructs the active site. For the single-domain class III Fic proteins, the αinh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins

The dynamic dimer structure of the chaperone Trigger Factor

The chaperone Trigger Factor (TF) from Escherichia coli forms a dimer at cellular concentrations. While the monomer structure of TF is well known, the spatial arrangement of this dimeric chaperone storage form has remained unclear. Here, we determine its structure by a combination of high-resolution NMR spectroscopy and biophysical methods. TF forms a symmetric head-to-tail dimer, where the ribosome binding domain is in contact with the substrate binding domain, while the peptidyl-prolyl isomerase domain contributes only slightly to the dimer affinity. The dimer structure is highly dynamic, with the two ribosome binding domains populating a conformational ensemble in the center. These dynamics result from intermolecular in trans interactions of the TF client-binding site with the ribosome binding domain, which is conformationally frustrated in the absence of the ribosome. The avidity in the dimer structure explains how the dimeric state of TF can be monomerized also by weakly interacting clients

Disulfide-Bond-Induced Structural Frustration and Dynamic Disorder in a Peroxiredoxin from MAS NMR

Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive μs time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts

Regulation of chaperone function by coupled folding and oligomerization

The homotrimeric molecular chaperone Skp of Gram-negative bacteria facilitates the transport of outer membrane proteins across the periplasm. It has been unclear how its activity is modulated during its functional cycle. Here, we report an atomic-resolution characterization of the; Escherichia coli; Skp monomer-trimer transition. We find that the monomeric state of Skp is intrinsically disordered and that formation of the oligomerization interface initiates folding of the α-helical coiled-coil arms via a unique "stapling" mechanism, resulting in the formation of active trimeric Skp. Native client proteins contact all three Skp subunits simultaneously, and accordingly, their binding shifts the Skp population toward the active trimer. This activation mechanism is shown to be essential for; Salmonella; fitness in a mouse infection model. The coupled mechanism is a unique example of how an ATP-independent chaperone can modulate its activity as a function of the presence of client proteins

Menstrual cycle phase modulates emotional conflict processing in women with and without premenstrual syndrome (PMS): A pilot study

Background Premenstrual syndrome (PMS) is characterized by a cluster of psychological and somatic symptoms during the late luteal phase of the menstrual cycle that disappear after the onset of menses. Behavioral differences in emotional and cognitive processing have been reported in women with PMS, and it is of particular interest whether PMS affects the parallel execution of emotional and cognitive processing. Related to this is the question of how the performance of women with PMS relates to stress levels compared to women without PMS. Cortisol has been shown to affect emotional processing in general and it has also been shown that women with severe PMS have a particular cortisol profile. Methods We measured performance in an emotional conflict task and stress levels in women with PMS (n = 15) and women without PMS (n = 15) throughout their menstrual cycle. Results We found a significant increase (p = 0.001) in the mean reaction time for resolving emotional conflict from the follicular to the luteal cycle phase in all subjects. Only women with PMS demonstrated an increase in physiological and subjective stress measures during the luteal menstrual cycle phase. Conclusions Our findings suggest that the menstrual cycle modulates the integration of emotional and cognitive processing in all women. Preliminary data are supportive of the secondary hypothesis that stress levels are mediated by the menstrual cycle phase only in women with PMS. The presented evidence for menstrual cycle-specific differences in integrating emotional and cognitive information highlights the importance of controlling for menstrual cycle phase in studies that aim to elucidate the interplay of emotion and cognition