Signal CSP resonances of Methyl-13C-labeled pSTAT3 and uSTAT3 were

Signal transducer and activator of transcription (STAT) protein family are intracellular transcription factors involved in various cellular processes like immunity, proliferation, apoptosis and differentiation. There are seven members in STAT family of proteins of which STAT3, upon phosphorylation by cytokines and growth factors, form homo- or heterodimers and translocate to the nucleus where they act as transcription activators.



STAT3 shares similar domain architecture with its family members that includes an unstructured N-terminal domain (ND), coiled-coil domain (CCD), DNA-binding domain (DBD), linker domain (LD), Src homology 2 domain (SH2) and an unstructured transactivation domain (TAD) located at the C-terminus as depicted in figure 4. Loss of function mutation (I568F) in LD domain of STAT3 results in Hyperimmunoglobulinemia E syndrome (HIES) characterized by facial, dental and skeletal abnormalities. This study reports the use of NMR spectroscopy and site-directed mutagenesis to demonstrate the inter-domain conformational fluctuations resulting in allosteric communication that controls STAT3 function in disease-inducing mutations.

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Methods and Key findings:

A well-dispersed two-dimensional (2D) 13C-1H heteronuclear multiple quantum coherence (HMQC) spectrum of the 68 kDa construct of the unphosphorylated STAT3 (uSTAT3) core region (127-711) was obtained by labelling C?1 methyl group of all 35 Ile. Significant changes in chemical shift perturbation (CSP) of SH2 domain of uSTAT3 upon binding to either phosphorylated IL-6 receptor or p-Tyr region of another STAT molecule was evident along with surprising CSP in the LD domain of STAT3. This interesting result provides the initial confirmation of inter domain dynamics.


To further investigate the cross domain dynamics, CSP resonances of Methyl-13C-labeled pSTAT3 and uSTAT3 were analyzed for dimer formation. Temperature dependent monomer dissociation has also been characterized and confirmed by Van’t Hoff analysis. The results conclude that there exists a monomer-dimer equilibrium which gets disturbed upon increase in temperature.


13C single-quantum (SQ) Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments were performed to understand the dynamics of the hydrophobic core of STAT3 domains in ?s-ms timescales. The authors have quantified the effective transverse relaxation rate R2,eff for 7 Ile residues, representative for each of STAT3 domains


Ile Chi-2 (?2) rotameric calculations from 13C chemical shifts of hydrophobic core residues showed at least two significant sidechain rotameric conformations in equilibrium. Noteworthy change in the rotameric distribution of LD domain, in particular residue I568 upon binding pTyr-peptide at the SH2 domain strengthens the view of allosteric effect.


The CSP of I568F (LD domain), HIES associated mutant undergoes largest change in shift when SH2 domain is bound to pTyr-containing peptide. This result substantiates the functional importance of the mutation when compared with wild-type CSP data indicating structural dynamics in all the STAT3 domains. Isothermal titration calorimetry further confirmed the reduction in binding affinities of SH2 domain of STAT3 due to this mutation.  



1.    Recently advanced NMR methods have been performed by the authors to show the inter-domain conformational dynamics of STAT3.


2.    The authors have not probed into more mutational studies (other HIES mutations/ disease mutations) involving other STAT3 domains to gain detailed insights into the suggested molecular mechanism of cross-domain allostery.


Outcome of the study:

The study presents novel insights into STAT3 functional dynamics and regulation through inter-domain allosteric communications and elucidates how further functional changes, upon disease causing mutation in a distal site, can perturb the regulation of STAT3 pathway.



Studies on deducing allosteric regulatory mechanisms largely focus on static structures rather than dynamic motions. The three articles reviewed in this report have exploited the advancements in NMR spectroscopic techniques to study the structural fluctuations and conformational dynamics upon ligand binding complemented with sensitive fluorescence based binding studies. Thermodynamic fluctuations have been captured by ITC binding experiments. These currently advancing protein dynamics studies assist us to understand in great detail the mechanisms of dynamic allostery and conformational complexity in picosecond-nanosecond time-scales. We can extend these findings to other members of these protein families and postulate a general mechanism of allostery. Similar studies can be carried out to enhance our knowledge on effect of post-tanslational modifications on ligand binding. 


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