Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization

Kocyła, Anna; Krężel, Artur

doi:10.1039/c8cc06301j

Cited by 9 publications

(14 citation statements)

References 37 publications

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“…Although there is evidence that both CD4 and Lck can form homodimers when expressed alone 9,27,53 , our sFCCS measurements show that the CD4-Lck heterodimer dominates when they are co-expressed, consistent with earlier BRET measurements 9 . In HEK293T cells, Lck expression was reduced when it could not bind CD4, implying that the trafficking of Lck is linked to CD4 association.…”

Section: Discussionsupporting

confidence: 90%

The kinase occupancy of T-cell coreceptors reconsidered

Mørch

Schneider

Jenkins

et al. 2022

Preprint

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The sensitivity of the αβ T-cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αβ, which are expressed primarily by cells of the helper and cytotoxic T-cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor-kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4-Lck interaction was stoichiometric (~100%) and that the CD8αβ-Lck interaction was also substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor-Lck co-diffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to show that stoichiometric Lck coupling required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 out-competed CD8αβ for Lck. In T cells, TCR signaling induced CD4-Lck oligomerization but did not affect the high levels of CD4-Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.Significance statementCD4 and CD8αβ are archetypal coreceptor proteins that potently enhance T-cell antigen sensitivity but how they function is still debated. A fundamental question that remains incompletely resolved is: what fractions of the coreceptors bind the signal-initiating kinase, Lck? Using in vitro assays and non-invasive fluorescence fluctuation spectroscopy in live cells, we show that most coreceptors are occupied by Lck at the surface of live cells. The structural basis for important differences in the kinase occupancy of CD4 and CD8αβ is also identified. These results provide important context for refining current models of both TCR antigen recognition and cell fate decisions made during thymopoiesis.

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Section: Discussionsupporting

confidence: 90%

The kinase occupancy of T-cell coreceptors reconsidered

Mørch

Schneider

Jenkins

et al. 2022

Preprint

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“…The application of the PPI model to metal-involved interaction with no consideration of Zn 2+ concentration leads to serious errors, as in the case of Zn 2+ -mediated CD4-Lck interaction. The initially reported K d value (submicromolar) was overestimated, suggesting that the ZPPI is not physiologically relevant until proven to be much lower with the agreement of the identified picomolar range of labile Zn 2+ concentration [92].…”

Section: Box 3 Stability Of Zppismentioning

confidence: 90%

“…Upon hGH release from the pituitary gland, the availability of the hormone is strictly limited by intracellular labile Zn 2+ [91]. Also, in the immunological process of lymphocyte development and antigen-dependent activation of mature T cells, one of the first-ever defined Zn 2+ -mediated heterodimeric complexes, zinc clasp, brings lymphoid-specific, Src family protein-tyrosine kinase (Lck) kinase through cluster of differentiation 4 (CD4)/CD8α coreceptor binding to proximity to T cell receptor (TCR) [92,93]. Specificity and stability of heterocomplex formation are affected by structural and sequential CD4 molecular determinants [16,92].…”

Section: Screening Of Structural Protein Database For Functional Zppismentioning

confidence: 99%

“…Also, in the immunological process of lymphocyte development and antigen-dependent activation of mature T cells, one of the first-ever defined Zn 2+ -mediated heterodimeric complexes, zinc clasp, brings lymphoid-specific, Src family protein-tyrosine kinase (Lck) kinase through cluster of differentiation 4 (CD4)/CD8α coreceptor binding to proximity to T cell receptor (TCR) [92,93]. Specificity and stability of heterocomplex formation are affected by structural and sequential CD4 molecular determinants [16,92]. Moreover, the balance between CD4 and CD4 homodimers was indicated to modulate the threshold of T cell activation [93].…”

Section: Screening Of Structural Protein Database For Functional Zppismentioning

confidence: 99%

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Galvanization of Protein–Protein Interactions in a Dynamic Zinc Interactome

Kocyła

Tran

Krężel

2021

Trends in Biochemical Sciences

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The presence of Zn 2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn 2+ -dependent manner. Recent studies have demonstrated that dynamically changing Zn 2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn 2+ -involved protein-protein interactions (ZPPIs) are formed and dissociate and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to experimental obstacles. Here, we review recent research advances on the role of Zn 2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and experimental challenges required for the identification and investigation of ZPPIs. Protein-Protein Interactome and the Role of Metal IonsA complete picture of protein-protein interactions (PPIs), also known as the protein-protein interactome (see Glossary), is crucial to understand cell molecular machinery at the system biology level [1-3]. Identification of protein molecular complexes requires the description of various aspects, such as composition, component affinity, and lifetime with series of spatial and physicochemical properties describing the interface between proteins. Considering both the affinity and lifetime of proteins to interact, obligate and nonobligate complexes can be found, where the latter are divided into permanent (mostly strong, irreversible) and transient (reversible, frequently with medium affinity). Transient complexes are characterized by smaller interfaces, frequently dependent on specific recognition patterns, where the limited surface meets the requirement of constituents to fold and exist independently without aggregation [4,5]. Their interfaces contain more residues of higher polarity stabilized by salt bridges and hydrogen bonds with more chain turns to gain flexibility [6][7][8]. The specificity of such interactions is also elevated by the utilization of the same interface for multiple interactions across evolution, where the local environment has been affecting the fast adaptation of mutations [9,10].

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“…To date, only a few intermolecular binding sites have been examined for Zn(II) affinity. For example, intermolecular Zn(II) binding sites in CD4/CD8a co-receptors—Lck kinase complexes [ 3 , 51 , 52 ]—and a Rad50 homolog from P. furiosus have been investigated in terms of stability [ 15 ]. On the other hand, Zn(II) affinity comparison of a complex that contains intermolecular Zn(II) with complexes with intramolecular Zn(II) (e.g., zinc fingers) is definitely more difficult due to differences in stoichiometry and constants’ definition, but can be done by applying the competitivity index (CI) [ 9 , 53 ], as demonstrated in Table 2 .…”

Section: Discussionmentioning

confidence: 99%

Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation

Tran¹,

Padjasek²,

Krężel³

2022

IJMS

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The metal binding at protein–protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence–structure–stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein–protein interactions and illuminates their molecular basis.

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Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization

Cited by 9 publications

References 37 publications

The kinase occupancy of T-cell coreceptors reconsidered

The kinase occupancy of T-cell coreceptors reconsidered

Galvanization of Protein–Protein Interactions in a Dynamic Zinc Interactome

Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation

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