Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions

Abstract Amino acid substitutions at nonconserved protein positions can have noncanonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

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Substitutions at Non-Conserved Rheostat Positions Modulate Function by Re-Wiring Long-Range, Dynamic Interactions

10.1101/2020.07.02.185207 ◽

2020 ◽

Cited By ~ 1

Author(s):

Paul Campitelli ◽

Liskin Swint-Kruse ◽

S. Banu Ozkan

Keyword(s):

Dna Binding ◽

Long Range ◽

Protein Function ◽

Binding Domain ◽

Dna Binding Domain ◽

Dynamic Coupling ◽

Long Distance ◽

Modulate Function ◽

Functional Changes ◽

Wide Range

AbstractAmino acid substitutions at nonconserved protein positions can have non-canonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities (“DFI”) and dynamic coupling (“DCI”) for positions in the linker region of the lactose repressor protein (“LacI”). This region contains nonconserved positions for which substitutions alter DNA binding affinity. We first chose to study eleven substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA binding positions, and the degree of flexibility change correlated with experimentally-measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA binding positions in a manner that captured other experimentally-determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated almost no substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type LacI, the dynamic couplings between the DNA binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and non-canonical substitutions outcomes at nonconserved positions arise from re-wiring long-range communication among functionally-important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

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DNA-Binding Domain of DNA Ligase from the Thermophilic Archaeon Pyrococcus abyssi: Improving Long-Range PCR and Neutralization of Heparin’s Inhibitory Effect

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-015-1683-2 ◽

2015 ◽

Vol 176 (7) ◽

pp. 1859-1869 ◽

Cited By ~ 3

Author(s):

Igor P. Oscorbin ◽

U. A. Boyarskikh ◽

A. I. Zakabunin ◽

E. A. Khrapov ◽

M. L. Filipenko

Keyword(s):

Dna Binding ◽

Long Range ◽

Inhibitory Effect ◽

Binding Domain ◽

Dna Ligase ◽

Dna Binding Domain ◽

Thermophilic Archaeon ◽

Long Range Pcr ◽

Pyrococcus Abyssi

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Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain

ACS Nano ◽

10.1021/acsnano.6b05938 ◽

2016 ◽

Vol 11 (1) ◽

pp. 144-152 ◽

Cited By ~ 23

Author(s):

Armando Hernandez-Garcia ◽

Nicole A. Estrich ◽

Marc W. T. Werten ◽

Johan R. C. Van Der Maarel ◽

Thomas H. LaBean ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

Dna Templates ◽

Protein Polymer ◽

Wide Range

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Long-Range Repression by Multiple Polycomb Group (PcG) Proteins Targeted by Fusion to a Defined DNA-Binding Domain in Drosophila

Genetics ◽

10.1093/genetics/158.1.291 ◽

2001 ◽

Vol 158 (1) ◽

pp. 291-307

Author(s):

Robin R Roseman ◽

Kelly Morgan ◽

Daniel R Mallin ◽

Rachel Roberson ◽

Timothy J Parnell ◽

...

Keyword(s):

Dna Binding ◽

Long Range ◽

Binding Domain ◽

Polycomb Group ◽

Dna Binding Domain ◽

Interaction Domain ◽

Bona Fide ◽

Biochemical Analyses ◽

Chromosomal Loci

Abstract A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.

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Molecular Dynamics Simulation in Solvent of the Bacteriophage 434 cI Repressor Protein DNA Binding Domain Amino Acids (R1–69) in Complex with its Cognate Operator (OR1) DNA Sequence

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.1999.10508336 ◽

1999 ◽

Vol 17 (1) ◽

pp. 1-17 ◽

Cited By ~ 2

Author(s):

Lester F. Harris ◽

Michael R. Sullivan ◽

Pamela D. Popken-Harris

Keyword(s):

Molecular Dynamics ◽

Amino Acids ◽

Molecular Dynamics Simulation ◽

Dna Binding ◽

Dna Sequence ◽

Binding Domain ◽

Dynamics Simulation ◽

Dna Binding Domain ◽

Repressor Protein ◽

Bacteriophage 434

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A Quantitative Systems Approach to Define Novel Effects of Tumour p53 Mutations on Binding Oncoprotein MDM2

International Journal of Molecular Sciences ◽

10.3390/ijms23010053 ◽

2021 ◽

Vol 23 (1) ◽

pp. 53

Author(s):

Manuel Fuentes ◽

Sanjeeva Srivastava ◽

Angela M. Gronenborn ◽

Joshua LaBaer

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Hot Spot ◽

Point Mutations ◽

Protein Microarrays ◽

Binding Domain ◽

Structural Domain ◽

Dna Binding Domain ◽

Label Free ◽

Wide Range

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.

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“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein

10.1101/2020.11.17.386979 ◽

2020 ◽

Author(s):

Leonidas E. Bantis ◽

Daniel J. Parente ◽

Aron W. Fenton ◽

Liskin Swint-Kruse

Keyword(s):

Phylogenetic Analyses ◽

Functional Parameter ◽

Molecular Biophysics ◽

Sequence Alignments ◽

Multiple Sequence ◽

Repressor Protein ◽

Functional Changes ◽

Lactose Repressor ◽

Wide Range ◽

Multivariable Analyses

AbstractAmino acid variation at “rheostat” positions provides opportunity to modulate various aspects of protein function – such as binding affinity or allosteric coupling – across a wide range. Previously a subclass of “multiplex” rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratory’s dataset of ∼4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) “single” rheostat positions that modulate only one functional parameter, (ii) “toggle” positions that follow textbook substitution rules, and (iii) “neutral” positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses – such as ConSurf – could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites.

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The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

Pharmaceuticals ◽

10.3390/ph6080960 ◽

2013 ◽

Vol 6 (8) ◽

pp. 960-987 ◽

Cited By ~ 9

Author(s):

Axel Weber ◽

Corina Borghouts ◽

Christian Brendel ◽

Richard Moriggl ◽

Natalia Delis ◽

...

Keyword(s):

Dna Binding ◽

Tumor Cells ◽

Human Tumor ◽

Binding Domain ◽

Dna Binding Domain ◽

Protein Activity ◽

Intracellular Degradation ◽

Wide Range ◽

Peptide Aptamer ◽

Prostate Tumor Cells

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment.

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