Approaching infinite affinity through engineering of peptide–protein interaction

Much of life’s complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide–protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen–deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide–protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.

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SUMO in the mammalian response to DNA damage

Biochemical Society Transactions ◽

10.1042/bst0380092 ◽

2010 ◽

Vol 38 (1) ◽

pp. 92-97 ◽

Cited By ~ 19

Author(s):

Joanna R. Morris

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Protein Function ◽

Mammalian Cells ◽

Cancer Susceptibility ◽

Susceptibility Gene ◽

Genotoxic Stress ◽

Breast Cancer Susceptibility ◽

Model Organisms ◽

Breast Cancer Susceptibility Gene

Modification by SUMOs (small ubiquitin-related modifiers) is largely transient and considered to alter protein function through altered protein–protein interactions. These modifications are significant regulators of the response to DNA damage in eukaryotic model organisms and SUMOylation affects a large number of proteins in mammalian cells, including several proteins involved in the response to genomic lesions [Golebiowski, Matic, Tatham, Cole, Yin, Nakamura, Cox, Barton, Mann and Hay (2009) Sci. Signaling 2, ra24]. Furthermore, recent work [Morris, Boutell, Keppler, Densham, Weekes, Alamshah, Butler, Galanty, Pangon, Kiuchi, Ng and Solomon (2009) Nature 462, 886–890; Galanty, Belotserkovskaya, Coates, Polo, Miller and Jackson (2009) Nature 462, 935–939] has revealed the involvement of the SUMO cascade in the BRCA1 (breast-cancer susceptibility gene 1) pathway response after DNA damage. The present review examines roles described for the SUMO pathway in the way mammalian cells respond to genotoxic stress.

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Apoptosis and development

Essays in Biochemistry ◽

10.1042/bse0390011 ◽

2003 ◽

Vol 39 ◽

pp. 11-24 ◽

Cited By ~ 6

Author(s):

Justin V McCarthy

Keyword(s):

Drosophila Melanogaster ◽

Mammalian Cells ◽

Cell Number ◽

Model Organisms ◽

Biological Processes ◽

Nematode Caenorhabditis Elegans ◽

Apoptotic Pathway ◽

Genetic Pathways ◽

Evolutionarily Conserved ◽

Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

Microorganisms ◽

10.3390/microorganisms9061323 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1323

Author(s):

Etai Boichis ◽

Nadejda Sigal ◽

Ilya Borovok ◽

Anat A. Herskovits

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Mammalian Cells ◽

Protein Pair ◽

Growth Conditions ◽

Wall Structure ◽

Antibiotic Resistant ◽

Extracellular Milieu ◽

Virion Release ◽

Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

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Molecular motors--a paradigm for mutant analysis

Journal of Cell Science ◽

10.1242/jcs.113.8.1311 ◽

2000 ◽

Vol 113 (8) ◽

pp. 1311-1318 ◽

Cited By ~ 1

Author(s):

S.A. Endow

Keyword(s):

Molecular Motors ◽

Protein Function ◽

Rational Design ◽

Atp Hydrolysis ◽

Unique Property ◽

Genetic Screens ◽

Nucleotide Hydrolysis ◽

Mutant Analysis ◽

Motor Activities ◽

Natural Variants

Molecular motors perform essential functions in the cell and have the potential to provide insights into the basis of many important processes. A unique property of molecular motors is their ability to convert energy from ATP hydrolysis into work, enabling the motors to bind to and move along cytoskeletal filaments. The mechanism of energy conversion by molecular motors is not yet understood and may lead to the discovery of new biophysical principles. Mutant analysis could provide valuable information, but it is not obvious how to obtain mutants that are informative for study. The analysis presented here points out several strategies for obtaining mutants by selection from molecular or genetic screens, or by rational design. Mutants that are expected to provide important information about the motor mechanism include ATPase mutants, which interfere with the nucleotide hydrolysis cycle, and uncoupling mutants, which unlink basic motor activities and reveal their interdependence. Natural variants can also be exploited to provide unexpected information about motor function. This general approach to uncovering protein function by analysis of informative mutants is applicable not only to molecular motors, but to other proteins of interest.

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Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

Scientific Reports ◽

10.1038/srep20331 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 23

Author(s):

Encarnación Medina-Carmona ◽

Rogelio J. Palomino-Morales ◽

Julian E. Fuchs ◽

Esperanza Padín-Gonzalez ◽

Noel Mesa-Torres ◽

...

Keyword(s):

Protein Dynamics ◽

Protein Function ◽

Conformational Dynamics ◽

Loss Of Function ◽

Quinone Oxidoreductase ◽

Protein Levels ◽

Terminal Domain ◽

Directional Preference

Abstract Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S and to develop new pharmacological therapies to rescue this function.

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Intermolecular correlations are necessary to explain diffuse scattering from protein crystals

IUCrJ ◽

10.1107/s2052252518001124 ◽

2018 ◽

Vol 5 (2) ◽

pp. 211-222 ◽

Cited By ~ 9

Author(s):

Ariana Peck ◽

Frédéric Poitevin ◽

Thomas J. Lane

Keyword(s):

Rigid Body ◽

Conformational Changes ◽

Protein Function ◽

Diffuse Scattering ◽

Conformational Dynamics ◽

Protein Crystals ◽

Biological Unit ◽

Long Range Correlations ◽

Correlated Motions ◽

Potential Applications

Conformational changes drive protein function, including catalysis, allostery and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work has challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid-body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signal reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid-body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.

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Autoamplification and competition drive symmetry breaking: Initiation of centriole duplication by the PLK4-STIL network

10.1101/430264 ◽

2018 ◽

Author(s):

Marcin Leda ◽

Andrew J. Holland ◽

Andrew B. Goryachev

Keyword(s):

Symmetry Breaking ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Break Symmetry ◽

Biophysical Model ◽

Model Organisms ◽

Centriole Duplication ◽

Mother Centriole ◽

Autocatalytic Activation

SummarySymmetry breaking, a central principle of physics, has been hailed as the driver of self-organization in biological systems in general and biogenesis of cellular organelles in particular, but the molecular mechanisms of symmetry breaking only begin to become understood. Centrioles, the structural cores of centrosomes and cilia, must duplicate every cell cycle to ensure their faithful inheritance through cellular divisions. Work in model organisms identified conserved proteins required for centriole duplication and found that altering their abundance affects centriole number. However, the biophysical principles that ensure that, under physiological conditions, only a single procentriole is produced on each mother centriole remain enigmatic. Here we propose a mechanistic biophysical model for the initiation of procentriole formation in mammalian cells. We posit that interactions between the master regulatory kinase PLK4 and its activator-substrate STIL form the basis of the procentriole initiation network. The model faithfully recapitulates the experimentally observed transition from PLK4 uniformly distributed around the mother centriole, the “ring”, to a unique PLK4 focus, the “spot”, that triggers the assembly of a new procentriole. This symmetry breaking requires a dual positive feedback based on autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4-STIL complexes by phosphorylated STIL. We find that, contrary to previous proposals,in situdegradation of active PLK4 is insufficient to break symmetry. Instead, the model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes explains both the instability of the PLK4 ring and formation of the unique PLK4 spot. In the model, strong competition at physiologically normal parameters robustly produces a single procentriole, while increasing overexpression of PLK4 and STIL weakens the competition and causes progressive addition of procentrioles in agreement with experimental observations.

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iProteinDB: an integrative database of Drosophila post-translational modifications

10.1101/386268 ◽

2018 ◽

Cited By ~ 2

Author(s):

Yanhui Hu ◽

Richelle Sopko ◽

Verena Chung ◽

Romain A. Studer ◽

Sean D. Landry ◽

...

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Large Scale ◽

Model Organisms ◽

General Strategy ◽

Post Translational Modification ◽

Post Translational Modifications ◽

Functional Sites ◽

Evolutionarily Conserved ◽

And Function

AbstractPost-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing stability, protein interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, commonly serine, threonine and tyrosine. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that many phosphorylation sites may be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites with regards to regulation and function. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus laevis, Danio rerio, and Caenorhabditis elegans.

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Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.008422 ◽

2019 ◽

Vol 294 (28) ◽

pp. 10877-10885 ◽

Cited By ~ 2

Author(s):

Da-Wei Lin ◽

Benjamin P. Chung ◽

Jia-Wei Huang ◽

Xiaorong Wang ◽

Lan Huang ◽

...

Keyword(s):

Protein Function ◽

Mammalian Cells ◽

Gene Locus ◽

End Joining ◽

High Quality ◽

Physiological Conditions ◽

Protein Tracking ◽

Endogenous Proteins ◽

And Behavior ◽

Fluorescence Tags

Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His6FLAG, His6-Biotin-TEV-RGSHis6, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.

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Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1091 ◽

2014 ◽

Vol 25 (22) ◽

pp. 3610-3618 ◽

Cited By ~ 34

Author(s):

Robert Mahen ◽

Birgit Koch ◽

Malte Wachsmuth ◽

Antonio Z. Politi ◽

Alexis Perez-Gonzalez ◽

...

Keyword(s):

Single Molecule ◽

Protein Function ◽

Mammalian Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Quantitative Imaging ◽

Live Cells ◽

Single Molecule Spectroscopy ◽

Mitotic Kinase ◽

Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

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