Intrinsically disordered caldesmon binds calmodulin via the “buttons on a string” mechanism

String Model ◽

Electrostatic Attraction ◽

Disordered Proteins ◽

Wild Type ◽

Tryptophan Residues ◽

Specific Packing ◽

We show here that chicken gizzard caldesmon (CaD) and its C-terminal domain (residues 636-771, CaD136) are intrinsically disordered proteins. The computational and experimental analyses of the wild type CaD136 and series of its single tryptophan mutants (W674A, W707A, and W737A) and a double tryptophan mutant (W674A/W707A) suggested that although the interaction of CaD136 with calmodulin (CaM) can be driven by the non-specific electrostatic attraction between these oppositely charged molecules, the specificity of CaD136-CaM binding is likely to be determined by the specific packing of important CaD136 tryptophan residues at the CaD136-CaM interface. It is suggested that this interaction can be described as the “buttons on a charged string” model, where the electrostatic attraction between the intrinsically disordered CaD136 and the CaM is solidified in a “snapping buttons” manner by specific packing of the CaD136 “pliable buttons” (which are the short segments of fluctuating local structure condensed around the tryptophan residues) at the CaD136-CaM interface. Our data also show that all three “buttons” are important for binding, since mutation of any of the tryptophans affects CaD136-CaM binding and since CaD136 remains CaM-buttoned even when two of the three tryptophans are mutated to alanines.

Does intrinsically disordered caldesmon bind calmodulin via the “buttons on a string” mechanism?

10.7287/peerj.preprints.1261 ◽

2015 ◽

Author(s):

Sergei E Permyakov ◽

Eugene A Permyakov ◽

Vladimir N Uversky

Keyword(s):

Local Structure ◽

String Model ◽

Electrostatic Attraction ◽

Disordered Proteins ◽

Wild Type ◽

Tryptophan Residues ◽

Specific Packing ◽

We show here that chicken gizzard caldesmon (CaD) and its C-terminal domain (residues 636-771, CaD136) are intrinsically disordered proteins. The computational and experimental analyses of the wild type CaD136 and series of its single tryptophan mutants (W674A, W707A, and W737A) and a double tryptophan mutant (W674A/W707A) suggested that although the interaction of CaD136 with calmodulin (CaM) can be driven by the non-specific electrostatic attraction between these oppositely charged molecules, the specificity of CaD136-CaM binding is likely to be determined by the specific packing of important CaD136 tryptophan residues at the CaD136-CaM interface. It is suggested that this interaction can be described as the “buttons on a charged string” model, where the electrostatic attraction between the intrinsically disordered CaD136 and the CaM is solidified in a “snapping buttons” manner by specific packing of the CaD136 “pliable buttons” (which are the short segments of fluctuating local structure condensed around the tryptophan residues) at the CaD136-CaM interface. Our data also show that all three “buttons” are important for binding, since mutation of any of the tryptophans affects CaD136-CaM binding and since CaD136 remains CaM-buttoned even when two of the three tryptophans are mutated to alanines.

Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins

10.26434/chemrxiv.7312277.v1 ◽

2018 ◽

Cited By ~ 2

Author(s):

Rebecca Beveridge ◽

Lukasz Migas ◽

Rahul Das ◽

Rohit Pappu ◽

Richard Kriwacki ◽

...

Keyword(s):

Mass Spectrometry ◽

Ion Mobility ◽

Disordered Proteins ◽

Ion Mobility Mass Spectrometry ◽

Conformational Heterogeneity ◽

Wild Type ◽

Charged Residues ◽

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion Mobility-Mass Spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27<sup>Kip1</sup>. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, k14 and k56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (k31) and k14 where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, k56 with linear segregation of oppositely charged residues, leads to limited conformational heterogeneity and a narrow distribution of charged states. Molecular dynamics simulations demonstrate that the interplay between chain solvation and intra-chain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.

Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins

10.26434/chemrxiv.7312277 ◽

2018 ◽

Author(s):

Rebecca Beveridge ◽

Lukasz Migas ◽

Rahul Das ◽

Rohit Pappu ◽

Richard Kriwacki ◽

...

Keyword(s):

Mass Spectrometry ◽

Ion Mobility ◽

Disordered Proteins ◽

Ion Mobility Mass Spectrometry ◽

Conformational Heterogeneity ◽

Wild Type ◽

Charged Residues ◽

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion Mobility-Mass Spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27<sup>Kip1</sup>. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, k14 and k56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (k31) and k14 where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, k56 with linear segregation of oppositely charged residues, leads to limited conformational heterogeneity and a narrow distribution of charged states. Molecular dynamics simulations demonstrate that the interplay between chain solvation and intra-chain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.

A Family of Small Intrinsically Disordered Proteins Involved in Flagellum-Dependent Motility inSalmonella enterica

Journal of Bacteriology ◽

10.1128/jb.00415-18 ◽

2018 ◽

Vol 201 (2) ◽

Cited By ~ 2

Author(s):

Tamiko Oguri ◽

Youjeong Kwon ◽

Jerry K. K. Woo ◽

Gerd Prehna ◽

Hyun Lee ◽

...

Keyword(s):

Expression Profiles ◽

Functional Characterization ◽

Disordered Proteins ◽

Wild Type ◽

Content Type ◽

Cellular Pathway ◽

Small Proteins ◽

Wide Range

ABSTRACTBy screening a collection ofSalmonellamutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF,STM14_1829, andyciG) required for wild-type flagellum-dependent swimming and swarming motility. TheymdF,STM14_1829, andyciGgenes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829mutant due to the downregulation of theflhDCoperon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829mutant, suggesting the indirect repression of theflhDCoperon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, includingEscherichia coliandPseudomonas aeruginosa. We showed that the inactivation of STM14_1829 homologs inE. coliandP. aeruginosaalso alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCEThis study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility inSalmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins

Journal of the American Chemical Society ◽

10.1021/jacs.8b13483 ◽

2019 ◽

Vol 141 (12) ◽

pp. 4908-4918 ◽

Cited By ~ 21

Author(s):

Rebecca Beveridge ◽

Lukasz G. Migas ◽

Rahul K. Das ◽

Rohit V. Pappu ◽

Richard W. Kriwacki ◽

...

Keyword(s):

Mass Spectrometry ◽

Ion Mobility ◽

Disordered Proteins ◽

Ion Mobility Mass Spectrometry ◽

Charged Residues ◽

The Impact ◽

Hierarchical Ensembles of Intrinsically Disordered Proteins at Atomic Resolution in Molecular Dynamics Simulations

10.1101/731133 ◽

2019 ◽

Author(s):

Lisa M. Pietrek ◽

Lukas S. Stelzl ◽

Gerhard Hummer

Keyword(s):

Molecular Dynamics ◽

Local Structure ◽

High Performance ◽

Large Fraction ◽

Conformational Dynamics ◽

Full Length ◽

Atomic Resolution ◽

Disordered Proteins ◽

Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) constitute a large fraction of the human proteome and are critical in the regulation of cellular processes. A detailed understanding of the conformational dynamics of IDPs could help to elucidate their roles in health and disease. However the inherent flexibility of IDPs makes structural studies and their interpretation challenging. Molecular dynamics (MD) simulations could address this challenge in principle, but inaccuracies in the simulation models and the need for long simulations have stymied progress. To overcome these limitations, we adopt an hierarchical approach that builds on the “flexible meccano” model of Bernadó et al. (J. Am. Chem. Soc. 2005, 127, 17968-17969). First, we exhaustively sample small IDP fragments in all-atom simulations to capture local structure. Then, we assemble the fragments into full-length IDPs to explore the stereochemically possible global structures of IDPs. The resulting ensembles of three-dimensional structures of full-length IDPs are highly diverse, much more so than in standard MD simulation. For the paradigmatic IDP α-synuclein, our ensemble captures both local structure, as probed by nuclear magnetic resonance (NMR) spectroscopy, and its overall dimension, as obtained from small-angle X-ray scattering (SAXS) in solution. By generating representative and meaningful starting ensembles, we can begin to exploit the massive parallelism afforded by current and future high-performance computing resources for atomic-resolution characterization of IDPs.

Raman Evidence of p53-DBD Disorder Decrease upon Interaction with the Anticancer Protein Azurin

International Journal of Molecular Sciences ◽

10.3390/ijms20123078 ◽

2019 ◽

Vol 20 (12) ◽

pp. 3078 ◽

Cited By ~ 1

Author(s):

Sara Signorelli ◽

Salvatore Cannistraro ◽

Anna Rita Bizzarri

Keyword(s):

Principal Component ◽

Intrinsically Disordered Protein ◽

Random Coil ◽

Disordered Proteins ◽

Conformational Heterogeneity ◽

Tryptophan Residues ◽

Α Helix ◽

Β Sheet

Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa. The Raman spectra of the DBD and Azurin, isolated in solution or forming a complex, were analyzed by a combined analysis based on peak inspection, band convolution, and principal component analysis (PCA). In particular, our attention was focused on the Raman peaks of Tyrosine and Tryptophan residues, which are diagnostic markers of protein side chain environment, and on the Amide I band, of which the deconvolution allows us to extract information about α-helix, β-sheet, and random coil contents. The results show an increase of the secondary structure content of DBD concomitantly with a decrease of its conformational heterogeneity upon its binding to Azurin. These findings suggest an Azurin-induced conformational change of DBD structure with possible implications for p53 functionality.

Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1304749110 ◽

2013 ◽

Vol 110 (33) ◽

pp. 13392-13397 ◽

Cited By ~ 388

Author(s):

R. K. Das ◽

R. V. Pappu

Keyword(s):

Disordered Proteins ◽

Linear Sequence ◽

Charged Residues ◽

Sequence Distributions ◽

Salt-bridge Dynamics in Intrinsically Disordered Proteins: A trade-off between electrostatic interactions and structural flexibility

10.1101/220392 ◽

2017 ◽

Author(s):

Sankar Basu ◽

Parbati Biswas

Keyword(s):

Electrostatic Interactions ◽

Salt Bridge ◽

Disordered Proteins ◽

Salt Bridges ◽

Trade Off ◽

Local Rigidity ◽

Wide Range ◽

AbstractIntrinsically Disordered Proteins (IDPs) are enriched in charged and polar residues; and, therefore, electrostatic interactions play a predominant role in their dynamics. In order to remain multi-functional and exhibit their characteristic binding promiscuity, they need to retain considerable dynamic flexibility. At the same time, they also need to accommodate a large number of oppositely charged residues, which eventually lead to the formation of salt-bridges, imparting local rigidity. The formation of salt-bridges therefore oppose the desired dynamic flexibility. Hence, there appears to be a meticulous trade-off between the two mechanisms which the current study attempts to unravel. With this objective, we identify and analyze salt-bridges, both as isolated as well as composite ionic bond motifs, in the molecular dynamic trajectories of a set of appropriately chosen IDPs. Time evolved structural properties of these salt-bridges like persistence, associated secondary structural ′order-disorder′ transitions, correlated atomic movements, contribution in the overall electrostatic balance of the proteins have been studied in necessary detail. The results suggest that the key to maintain such a trade-off over time is the continuous formation and dissolution of salt-bridges with a wide range of persistence. Also, the continuous dynamic interchange of charged-atom-pairs (coming from a variety of oppositely charged side-chains) in the transient ionic bonds supports a model of dynamic flexibility concomitant with the well characterized stochastic conformational switching in these proteins. The results and conclusions should facilitate the future design of salt-bridges as a mean to further explore the disordered-globular interface in proteins.