Biology subjects

Prestel, A.

Publications and source records attributed to Prestel, A..

14 recordsLinked to original sources

bioRxiv2026

Cooperativity, dynamics, and the free-energy surfaces of charge-patterned IDPs

The free-energy surfaces that underlie the conformational distributions of intrinsically disordered proteins (IDPs) are shallow and lack the deep minima characteristic of stable, folded structures. However, even in the absence of secondary or tertiary structure, sequence patterning can lead to conformational preferences and changes in chain dimensions as a function of solution conditions. While patterning effects have received extensive attention from simulation and theory, there is little corresponding data from experiment. Here we investigate the impact of charge patterning on chain dimensions and dynamics in a set of specifically designed polyampholytic IDP variants across the natural range of charge segregation with single-molecule FRET, nanosecond fluorescence correlation, circular dichroism, and NMR spectroscopy. We find that the conformational ensembles and their cooperative response to salt concentration show prominent and systematic dependencies on charge patterning, and to some extent on residue type. In contrast, the chain dynamics remain in the tens-of-nanosecond range, consistent with the absence of pronounced free-energy barriers. In close combination with molecular simulations, we show how the concept of susceptibility can be used to quantify cooperativity in the absence of barriers and relate it to the shallow free-energy surfaces of IDPs.

biophysics↗

bioRxiv2024

The SH Protein of Mumps Virus is a Druggable Pentameric Viroporin

Viral infections are on the rise and drugs targeting viral proteins are needed. Viroporins constitute a growing group of virus-encoded transmembrane oligomeric proteins that allow passage of small molecules across the membrane. Despite sparsity in viroporin structures, recent work has revealed diversity in both the number of transmembrane helices and oligomeric states. Here we provide evidence that the small hydrophobic protein (SH) from mumps virus is a pentameric viroporin. From extensive biophysical data, a HADDOCK model of full-length SH shows its intracellular C-terminal region to form an extended structure crucial to stabilization of the pentamer. Heterologous expression of wild type SH and variants in Xenopus laevis oocytes reveals the viroporin as a chloride channel, facilitated by conserved hydroxyl-carrying residues lining the pore. The channel function of SH is inhibited by the small-molecule BIT225, highlighting the potential for antiviral targeting through SH.

biophysics↗

bioRxiv2024

Responses to ligand binding in the bacterial DNA sliding clamp beta-clamp manifest in dynamic allosteric effects

The homo-dimeric, ring-shaped bacterial DNA sliding clamp, {beta}-clamp, is a central hub in DNA replication and repair. It interacts with a plethora of proteins via short linear motifs, which bind to the same hydrophobic binding pocket on {beta}-clamp. Although the structure, functions and interactions of {beta}-clamp have been amply studied, less focus has been on understanding its dynamics and how this is influenced by ligand binding. In this work, we have made a backbone nuclear magnetic resonance (NMR) assignment of the 83 kDa dimeric {beta}-clamp and used NMR in combination with hydrogen-deuterium exchange mass spectrometry to scrutinize the dynamics of {beta}-clamp and how different ligands affect this. We found that binding of a small peptide from the polymerase III subunit affects the dynamics and stability of {beta}-clamp. This effect not only appears locally around the binding pocket, but also globally through dynamic allosteric connections to distant regions of the protein, including the dimer interface. This dissipated dynamic effect is likely a consequence of the binding pocket architecture and may reflect a common mechanism of structural plasticity, where different ligands impose differential responses in the structure and dynamics of {beta}-clamp. HighlightsO_LINMR spectroscopy of {beta}-clamp revealed its inherent dynamics C_LIO_LIA peptide ligand from polymerase III stabilizes {beta}-clamp from global unfolding C_LIO_LIPeptide binding causes allosteric effects that manifest in changes in dynamics C_LIO_LIDynamic changes occur distant to the binding pocket in the dimer interface C_LIO_LIAllostery may be a mechanism for differential responses to interaction partners C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/600769v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1a2fa22org.highwire.dtl.DTLVardef@d40d98org.highwire.dtl.DTLVardef@1c87aaforg.highwire.dtl.DTLVardef@ee8462_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

A suicidal and extensively disordered luciferase with a bright luminescence

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 [A] and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase. Significance statementEnzymes are typically characterized by an overall globular structure with a hydrophobic core and a defined cavity for binding of substrate, containing the active site amino acid residues. Gaussia Luciferase is a widely used luminescent reporter with a very strong, albeit short-lived, flash of light due to rapid auto-inactivation. We show, using solution NMR, that while this luciferase shows some secondary structure elements held together by disulfide bonds this highly unusual enzyme is extensively disordered with essentially no hydrophobic core. Although the enzymatic mechanism remains unknown, we have identified two essential arginine residues but, in the structure, these do not point into a common active site. In spite of this, the enzyme has high substrate specificity suggesting that it undergoes major structural rearrangements upon binding of substrate.

biochemistry↗

bioRxiv2023

DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles.

biophysics↗

bioRxiv2023

Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known. Using NMR spectroscopy, in vivo assays, X-ray crystallography, calorimetry, and Monte Carlo simulations, an additional PCNA binding motif in Spd1, a PIP-box, is revealed. The two tandemly positioned, low affinity sites exchange rapidly on PCNA exploiting the same binding sites. Increasing or decreasing the binding affinity between Spd1 and PCNA through mutations of either motif compromised the ability of Spd1 to cause checkpoint activation in yeast. These results pinpoint a role for PCNA in Spd1-mediated checkpoint activation and suggest that its tandemly positioned short linear motifs create a neatly balanced competition-based system, involving PCNA, Spd1 and the small ribonucleotide reductase subunit, Suc22R2. Similar mechanisms may be relevant in other PCNA binding ligands where divergent binding motifs so far have gone under the PIP-box radar.

biophysics↗

bioRxiv2023

A flexible loop in the paxillin LIM3 domain mediates direct binding to integrin β3

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance and haemostasis, but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, focal adhesion assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of focal adhesion sites, with the cytoplasmic tails of the integrin {beta}1 and {beta}3 subunits. The binding interface resides in paxillins LIM3 domain, where based on the NMR structure and functional analyses a flexible, seven amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin {beta}3 compromises cell adhesion and motility. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin {beta}3 function.

cell biology↗

bioRxiv2022

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nano second and faster dynamics. Here we asked what effect this mutant and the two individual mutants L49I and I57V have on the slow conformational dynamics of CI2. We used 15N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1 {degrees}C. As the temperature is increased the population of the excited state decreases. Structural changes in the transition to the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The mutations in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the mutated residues and the well-ordered water molecules links subtle structural changes around the mutated residues to the region in the protein that experience slow conformational changes.

biophysics↗

bioRxiv2022

Distinction between small RNA-bound and free ARGONAUTE via an N-terminal protein-protein interaction site

ARGONAUTE (AGO) proteins bind to small non-coding RNAs to form RNA Induced Silencing Complexes (RISCs). In the RNA-bound state, AGO proteins are stable while RNA-free AGOs turn over rapidly. Molecular determinants unique to RNA-free AGO that allow its specific recognition and degradation remain unknown. Here, we show that a confined, linear region in Arabidopsis AGO1, the N-coil, is accessible to antibodies preferentially in the RNA-free state of AGO1. Reanalysis of hydrogen-deuterium exchange data on human Ago2 indicates similar structural flexibility of the N-coil depending on small RNA binding. Unloaded Arabidopsis AGO1 interacts with the autophagy cargo receptor ATI1 via direct contact to specific amino acid residues in the N-coil, and mutation of residues required for ATI1 interaction reduces the degradation rate of unloaded AGO1 in vivo. These results provide insight into the molecular basis for specific recognition and degradation of the RNA-free state of eukaryotic AGO proteins.

molecular biology↗

bioRxiv2022

Structural characterization of human tryptophanhydroxylase 2 reveals L-Phe as the superior regulatorydomain ligand relevant for serotonin biosynthesis

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in the biosynthesis of serotonin in the brain. Consequently, regulation of TPH2 is relevant for serotonin related diseases, yet, the regulatory mechanism of TPH2 is poorly understood and structural as well as dynamical insights are missing. Here, we use NMR spectroscopy to determine the structure of a 47 N-terminally truncated variant of the regulatory domain (RD) dimer of human TPH2 in complex with L-Phe, and show that L-Phe is the superior RD ligand compared to the natural substrate, L-Trp. Using cryo-EM we obtain a low-resolution structure of a similarly truncated variant of the complete tetrameric enzyme with dimerized RDs. The cryo-EM 2D class averages additionally indicate that the RDs are dynamic in the tetramer and likely exist in a monomer-dimer equilibrium. Our results provide structural information on the RD both as an isolated domain and in the TPH2 tetramer, which will facilitate future elucidation of TPH2s regulatory mechanism affecting serotonin regulation.

biophysics↗

bioRxiv2022

Deciphering the alphabet of disorder -- Glu and Asp act differently on local but not global properties

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences of glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing data base of experimentally verified IDPs (DisProt) and show that the glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while Glu/Asp support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity where glutamates can support increased helicity important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in e.g., transactivation domains and ion-binding.

biophysics↗

bioRxiv2021

Double mutant of chymotrypsin inhibitor 2 stabilized through increased conformational entropy

The conformational heterogeneity of a folded protein can affect both its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here we have examined whether changed native state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side-chains with molecular dynamics simulations to quantify the native state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: A reduction in conformational dynamics (and entropy) of the native state of L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

biophysics↗

bioRxiv2020

Dynamic NHE1-Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts expressing wildtype and mutant NHE1, we discovered multiple accessible states of this important complex existing in different NHE1:CaM stoichiometries and structures. We solved the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities were tunable by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding -helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation or mutating the first CaM-binding helix, whereas Ca2+-induced NHE1 activity was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results reveal the diversity of NHE1:CaM interactions and suggest that CaM may contribute to NHE1 dimerization. We propose that similar structural diversity is relevant to other CaM complexes.

cell biology↗

bioRxiv2018

The Arabidopsis autophagy cargo receptors ATI1 and ATI2 bind to ATG8 via intrinsically disordered regions and are post-translationally modified upon ATG8 interaction

Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8 interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8 Interacting Protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties consistent with a collapsed pre-molten globule-like conformation Interestingly, wild type ATI1 and ATI2 exist as distinct post-translationally modified forms. Specifically, different forms are detectable upon mutation of the AIM, suggesting that interaction of ATI1 and ATI2 to ATG8 is coupled to a change in their post-translational modification.

molecular biology↗