Antibody-protein binding and conformational changes: identifying allosteric signalling pathways to engineer a better effector response

The dynamic interaction of cells of the immune system with other cells, antigens and secreted factors determines the nature of an immune response. The response of individual cells is governed by the sequence of intracellular signalling events triggered following the association of cell surface molecules during cell-cell contact or the detection of soluble molecules of host or pathogen origin. In this review we will first outline the general principles of intracellular signal transduction. We will then describe the signalling pathways triggered following the recognition of antigen, as well as the detection of cytokines, and discuss how the signalling pathways activated regulate the effector response.

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Conformational Changes in Protein Binding Processes

Biophysical Journal ◽

10.1016/j.bpj.2013.11.3609 ◽

2014 ◽

Vol 106 (2) ◽

pp. 652a

Author(s):

Béla Voβ ◽

Helmut Grubmüller

Keyword(s):

Protein Binding ◽

Conformational Changes

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Molecular mechanism of multispecific recognition of Calmodulin through conformational changes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615949114 ◽

2017 ◽

Vol 114 (20) ◽

pp. E3927-E3934 ◽

Cited By ~ 18

Author(s):

Fei Liu ◽

Xiakun Chu ◽

H. Peter Lu ◽

Jin Wang

Keyword(s):

Protein Binding ◽

Conformational Change ◽

Molecular Mechanism ◽

Conformational Changes ◽

Electrostatic Interaction ◽

Hydrophobic Interaction ◽

High Specificity ◽

Binding Peptide ◽

Conformational Selection ◽

High Affinity

Calmodulin (CaM) is found to have the capability to bind multiple targets. Investigations on the association mechanism of CaM to its targets are crucial for understanding protein–protein binding and recognition. Here, we developed a structure-based model to explore the binding process between CaM and skMLCK binding peptide. We found the cooperation between nonnative electrostatic interaction and nonnative hydrophobic interaction plays an important role in nonspecific recognition between CaM and its target. We also found that the conserved hydrophobic anchors of skMLCK and binding patches of CaM are crucial for the transition from high affinity to high specificity. Furthermore, this association process involves simultaneously both local conformational change of CaM and global conformational changes of the skMLCK binding peptide. We found a landscape with a mixture of the atypical “induced fit,” the atypical “conformational selection,” and “simultaneously binding–folding,” depending on the synchronization of folding and binding. Finally, we extend our discussions on multispecific binding between CaM and its targets. These association characteristics proposed for CaM and skMLCK can provide insights into multispecific binding of CaM.

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Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Journal of Virology ◽

10.1128/jvi.78.15.8036-8046.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8036-8046 ◽

Cited By ~ 6

Author(s):

Gail Rocheleau ◽

Jessica Petrillo ◽

Laura Guogas ◽

Lee Gehrke

Keyword(s):

Coat Protein ◽

Protein Binding ◽

Mosaic Virus ◽

Conformational Changes ◽

Polyacrylamide Gel Electrophoresis ◽

Genetic Selection ◽

Alfalfa Mosaic Virus ◽

Rna Replication ◽

Viral Rna

ABSTRACT The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3′-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5′ hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3′-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3′ RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3′ hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.

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PROTEIN-RNA INTERACTIONS IN BACTERIAL RIBOSOMES

Acta Endocrinologica ◽

10.1530/acta.0.074s075 ◽

1973 ◽

Vol 74 (Suppl) ◽

pp. S75-S94 ◽

Cited By ~ 3

Author(s):

R. A. Garrett ◽

H. G. Wittmann

Keyword(s):

Protein Binding ◽

Structural Properties ◽

Conformational Changes ◽

Binding Sites ◽

Ribosomal Proteins ◽

Structural Factors ◽

Optimal Conditions ◽

Protein Binding Sites ◽

E Coli ◽

Experimental Approaches

ABSTRACT There are several ribosomal proteins which can bind specifically to the three ribosomal RNA's of E. coli. Some structural properties of these proteins and of the RNA are described together with the optimal conditions for binding. Evidence for conformational changes occurring in some proteins, and in the RNA's during binding, is also discussed. The partial localisation of protein binding sites on the 16S and 5S RNA's, and for one protein on the 23S RNA has been attained recently. However, relatively little is known about the regions of the protein which interact with the RNA. Although the nature of the specificity of the protein-RNA interactions is not yet understood, some experimental approaches which are in progress to elucidate the basis of this specificity are mentioned together with a discussion of the structural factors which may be important.

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An analysis of conformational changes upon RNA-protein binding

Proceedings of the 5th ACM Conference on Bioinformatics, Computational Biology, and Health Informatics - BCB '14 ◽

10.1145/2649387.2660790 ◽

2014 ◽

Cited By ~ 1

Author(s):

Kannan Sankar ◽

Rasna R. Walia ◽

Carla M. Mann ◽

Robert L. Jernigan ◽

Vasant G. Honavar ◽

...

Keyword(s):

Protein Binding ◽

Conformational Changes

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Differential BUM-HMM: a robust statistical modelling approach for detecting RNA flexibility changes in high-throughput structure probing data

10.1101/2020.07.30.229179 ◽

2020 ◽

Author(s):

Paolo Marangio ◽

Ka Ying Toby Law ◽

Guido Sanguinetti ◽

Sander Granneman

Keyword(s):

Protein Binding ◽

High Throughput ◽

Conformational Changes ◽

Binding Sites ◽

Rna Structure ◽

Structural Changes ◽

High Throughput Sequencing ◽

Structural Information ◽

Protein Binding Sites ◽

Structure Probing

Combining RNA structure probing with high-throughput sequencing technologies has greatly enhanced our ability to analyze RNA structure at transcriptome scale. However, the high level of noise and variability encountered in these data call for the development of computational tools that robustly extract RNA structural information. Here we present diffBUM-HMM, a noise-aware model that enables accurate detection of RNA flexibility and conformational changes from high-throughput RNA structure-probing data. DiffBUM-HMM is compatible with a wide variety of high-throughput RNA structure probing data, taking into consideration biological variation, sequence coverage and sequence representation biases. We demonstrate that, compared to the existing approaches, diffBUM-HMM displays higher sensitivity while calling virtually no false positives. DiffBUM-HMM analysis of ex vivo and in vivo Xist SHAPE-MaP data detected many more RNA structural differences, involving mostly single-stranded nucleotides located at or near protein-binding sites. Collectively, our analyses demonstrate the value of diffBUM-HMM for quantitatively detecting RNA structural changes and reinforce the notion that RNA structure probing is a very powerful tool for identifying protein-binding sites.

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High content imaging for monitoring signalling dynamics in single cells

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0169 ◽

2020 ◽

Vol 65 (4) ◽

pp. R91-R100

Author(s):

Kathryn L Garner

Keyword(s):

Single Cell ◽

Conformational Changes ◽

Protein Interactions ◽

Single Cells ◽

Cellular Heterogeneity ◽

Live Cells ◽

Signalling Pathways ◽

Protein Protein Interactions ◽

High Content Imaging ◽

Surface Receptors

All living cells are sensors of their environment: they sense signals, hormones, cytokines, and growth factors, among others. Binding of these signals to cell surface receptors initiates the transmission of messages along intracellular signalling pathways through protein–protein interactions, enzymatic modifications and conformational changes. Typically, the activation of signalling pathways are monitored in whole populations of cells, giving population average measures, often using experimental methods that destroy and homogenise the cell population. High content imaging is an automated, high-throughput fluorescence microscopy method that enables measurements of signal transduction pathways to be taken from live cells. It can be used to measure signalling dynamics, how the abundance of particular proteins of interest change over time, or to record how particular proteins move and change their localisation in response to a signal from their environment. Using this, and other single cell methods, it is becoming increasingly clear that cells are in fact very variable in their response to a given stimulus and in the quantities of cellular components they express, even in clonal (isogenic) cell lines. This review will discuss how high content imaging has contributed to our growing understanding of cellular heterogeneity. It will discuss how data generated has been combined with information theoretic approaches to quantify the amount of information transferred through noisy signalling pathways. Lastly, the relevance of heterogeneity to our understanding and treatment of disease will be considered, highlighting the importance of single cell measurements.

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Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

International Journal of Molecular Sciences ◽

10.3390/ijms22073517 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3517

Author(s):

Kwang-Im Oh ◽

Ae-Ree Lee ◽

Seo-Ree Choi ◽

Youyeon Go ◽

Kyoung-Seok Ryu ◽

...

Keyword(s):

Protein Binding ◽

Conformational Changes ◽

Global Minimum ◽

Global Search ◽

Relaxation Dispersion ◽

State Transitions ◽

Oscillatory Regime ◽

Cpmg Relaxation Dispersion ◽

Search Approach ◽

Dna Complex

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s−1, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.

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