Distal Loop Flexibility of a Regulatory Domain Modulates Dynamics and Activity of C-Terminal Src Kinase (Csk)

Barkho, Sulyman; Pierce, Levi C. T.; McGlone, Maria L.; Li, Sheng; Woods, Virgil L.; Walker, Ross C.; Adams, Joseph A.; Jennings, Patricia A.

doi:10.1371/journal.pcbi.1003188

Cited by 11 publications

(10 citation statements)

References 58 publications

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“…Instrument setup and operation were described previously (26). All frozen samples were thawed and run using the conditions determined during fragmentation optimization.…”

Section: Methodsmentioning

confidence: 99%

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Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1

Tamir¹,

Rotem‐Bamberger

Katz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)-and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29 (3):606-618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1-Bcl-2 complex. NAF-1 binds to both the pro-and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein-protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1-Bcl-2 complex in the regulation of apoptosis/ autophagy in cancer biology.ife requires a controlled balance of energy conversion and utilization. These critical processes are governed by an elaborate set of reactions involving numerous protein-protein interactions. Among them is the ability of organisms to control the recycling of high-energy compounds and to control cell proliferation. These processes are, at least in part, under the control of cell survival and programmed cell death (autophagic and apoptotic) processes. Misregulation of these processes leads to many diseases, including cancer. Among the key proteins involved in these processes are Bcl-2 (1, 2) and the more recently identified iron-sulfur (Fe-S) protein nutrient-deprivation autophagy factor-1 (NAF-1) (also known as Cisd2, Miner1, Eris, and Noxp70) (3-5).NAF-1 is important for human health and disease. Missplicing of NAF-1 causes Wolfram syndrome 2 (6). NAF-1 is also functionally linked to the regulation of autophagy in cancer, and aging (3-5, 7, 8). This protein is a member of the 2Fe-2S cluster NEET family. NAF-1 has a similar backbone fold and 3Cys-1His coordination of the 2Fe-2S cluster as found in the founding member of the NEET family, mitoNEET (mNT). NAF-1 differs from mNT in the distribution of charged and aromatic surface residues (9, 10). These differences alter the 3D shape and electrostatics of the surfaces of mNT and NAF-1, leading to interactions with distinct binding partners. In fact, recent work identifi...

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“…Instrument setup and operation were described previously (26). All frozen samples were thawed and run using the conditions determined during fragmentation optimization.…”

Section: Methodsmentioning

confidence: 99%

“…The quality of each peptide was monitored by individually examining each measured isotopic envelope spectrum for the entire time-course exchange. The deuterium content was calculated for each time point by using specialized software as previously described (26,30).…”

Section: Methodsmentioning

confidence: 99%

Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1

Tamir¹,

Rotem‐Bamberger

Katz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…To regulate the catalytic activity, such signals need to propagate from the protein’s regulatory sites to the ATP- and substrate-binding sites within the kinase domain. Although such communication has been explored previously 9 10 , the underlying mechanisms—and whether they lead to an allosteric interaction between the two binding sites—have not been completely elucidated, despite their importance for a better understanding of PTK regulation at a molecular level.…”

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confidence: 99%

A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

et al. 2015

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Protein tyrosine kinases are attractive drug targets because many human diseases are associated with deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phospho-peptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP competitive kinase inhibitors can affect their activity.

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“…Structural and dynamic studies of Csk have revealed the importance of maintaining optimal dynamics for efficient catalysis [ 26 – 28 ]. Perturbations to large global domain motions are likely to shift the exchange between solution state ensembles that can be detected via established solution methods [ 29 ].…”

Section: Resultsmentioning

confidence: 99%

“…(His) 6 tagged Csk and kdSrc were expressed and purified as previously described [ 28 ]. Site-Directed mutagenesis was performed according to standard protocols.…”

Section: Methodsmentioning

confidence: 99%

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

Barkho

Pierce

et al. 2015

PLoS ONE

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The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk.

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Distal Loop Flexibility of a Regulatory Domain Modulates Dynamics and Activity of C-Terminal Src Kinase (Csk)

Cited by 11 publications

References 58 publications

Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1

Integrated strategy reveals the protein interface between cancer targets Bcl-2 and NAF-1

A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

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