Abstract
Transcription factors must scan genomic DNA, recognize the cognate sequence of their control element(s), and bind tightly to them. The DNA recognition process is primarily carried out by their DNA binding domains (DBD), which interact with the cognate site with high affinity and more weakly with any other DNA sequence. DBDs are generally thought to bind to their cognate DNA without changing conformation (lock-and-key). Here, we used nuclear magnetic resonance and circular dichroism to investigate the interplay between DNA recognition and DBD conformation in the engrailed homeodomain (enHD), as a model case for the homeodomain family of eukaryotic DBDs. We found that the conformational ensemble of enHD is rather flexible and becomes gradually more disordered as ionic strength decreases following a Debye–Hückel’s dependence. Our analysis indicates that enHD’s response to ionic strength is mediated by a built-in electrostatic spring-loaded latch that operates as a conformational transducer. We also found that, at moderate ionic strengths, enHD changes conformation upon binding to cognate DNA. This change is of larger amplitude and somewhat orthogonal to the response to ionic strength. As a consequence, very high ionic strengths (e.g., 700 mM) block the electrostatic-spring-loaded latch and binding to cognate DNA becomes lock-and-key. However, the interplay between enHD conformation and cognate DNA binding is robust across a range of ionic strengths (i.e., 45 to 300 mM) that covers the physiologically-relevant conditions. Therefore, our results demonstrate the presence of a mechanism for the conformational control of cognate DNA recognition on a eukaryotic DBD. This mechanism can function as a signal transducer that locks the DBD in place upon encountering the cognate site during active DNA scanning. The electrostatic-spring-loaded latch of enHD can also enable the fine control of DNA recognition in response to transient changes in local ionic strength induced by variate physiological processes.
Highlights
Transcription factors control gene expression by binding to specific DNA control elements and thereby recruit or block the recruitment of the transcription machinery to the target gene [1]
On the engrailed homeodomain, as a model for eukaryotic DNA binding domain (DBD)
Homeodomains are members of the helix-turn-helix family of DBDs typically found in eukaryotic transcription factors that function as master regulators [36]
Summary
Transcription factors control gene expression by binding to specific DNA control elements and thereby recruit or block the recruitment of the transcription machinery to the target gene [1]. Nuclear magnetic resonance (NMR) structural studies of eukaryotic DBDs in absence of DNA have shown that, at the minimal ionic strength used in such NMR analyses, these proteins are marginally stable, or even unstable at room temperature [32–34]. We address these two puzzles by studying the interplay between the conformation of the DBD and its specific binding to the cognate DNA site. 2022, 23, 2412 special properties mentioned above for eukaryotic DBDs It recognizes a very s palindromic cognate sequence (TAATTA) [43] and binds to specific DNA by su inserting its C-terminal helix into the DNA major groove (Figure 1a). 1cHomabnidne1d5N1Hchaenmdi1c5aNl chemshift ical shift deviations observed in the HSQC spectrum with increasing NaCl concentration in a log dsecvailaet.ioTnhseosbidseervcheadininntihtreoHgeSnQCofsapregcitnriunme awnidthlyinsicnreearseinsigduNeasCalncdonscidenectrhaatiinonoixnygaelnogosfcgallue.taTmheate srideseidchuaeisnanrietrdoigsepnlaoyfeadrgasinbinlueeaannddlyresidnesprheseirdeus,esreasnpdecstiidveeclyh.aTinhoexsyidgeecnhoafingliuotnampaaitres rtehsaitdfuoersmarsealt dbisrpidlagyeesdinasthbeluneaatinvde reendHsDphsetrreusc,truersepeacrteivineldyi.cTahteedsiwdeitchhadionuibolne phaeiards ethdaat rfroorwmss,aalnt dbrtihdegierscihnatrhgeed naattoivme eisncHoDnnsetrcutecdtutroe tahre icnodrirceastpeodnwditnhgdCoαubblye haetahdinedlianreroowf tsh, eansdamtheeicroclohrarfgoerdreafteormenicsec.onnected to the corresponding Cα by a thin line of the same color for reference
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