Interestingly, the dimer assembles in such a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity. The structure shows that three out of the five substrate-binding sites are partially buried in the dimer, thus explaining why protein binding results in TF monomerization. We have taken advantage of recent advances in NMR spectroscopy and isotope labeling ( Huang and Kalodimos, 2017) to determine the atomic structure of dimeric TF. The large size of the dimeric TF (100 kDa) and its apparent dynamic nature has hindered determination of its structure. The interplay between substrate protein binding and chaperone oligomerization is likely to be used as a mechanism to modulate the energetics and kinetics of interaction in chaperone-substrate protein complexes, as for example in small heat shock proteins ( Eyles and Gierasch, 2010). Interestingly, our data showed that substrate protein binding causes TF to monomerize, thus indicating that the substrate-binding sites are occluded in dimeric TF. The structure revealed how the chaperone recognizes and engages the non-native protein and how it retains it in an unfolded state. We recently determined the atomic resolution structure of TF in complex with a non-native protein ( Saio et al., 2014). TF is also being widely used as a co-expression factor to improve the folding and yield of soluble proteins in biotechnology ( Uthailak et al., 2017). The trigger factor (TF) chaperone has several unique features ( Hoffmann et al., 2010 Ries et al., 2017 Wruck et al., 2018): (i) is the only ribosome-associated chaperone in bacteria (ii) with an estimated cellular concentration of ~50 μM ( Crooke et al., 1988) it is also the most abundant one (iii) in contrast to other oligomeric chaperones such as GroEL, SecB, and Hsp90 that form stable oligomers, TF undergoes a dynamic transition between a monomeric and a dimeric form (iv) TF functions both at the ribosome and in the cytosol: it binds, as a monomer, next to the exit channel at the ribosome to prevent the aggregation and premature folding of nascent polypeptides, while it functions as a dimer in the cytosol where is thought to assist in various processes in protein folding and biogenesis ( Agashe et al., 2004 Ferbitz et al., 2004 Haldar et al., 2017 Martinez-Hackert and Hendrickson, 2009 Oh et al., 2011 Ullers et al., 2007). Much less is known about how ATP-independent chaperones assist with protein folding ( Stull et al., 2016). Studies of ATP-dependent chaperones, such as the Hsp70 and GroEL systems, have shown how cycles of ATP binding, hydrolysis and nucleotide release can give rise to different conformational states that exhibit distinct affinities for the substrate protein ( Apetri and Horwich, 2008 Clare et al., 2012 Hayer-Hartl et al., 2016 Kampinga and Craig, 2010 Mayer and Bukau, 2005 Saibil et al., 2013 Sekhar et al., 2016 Zhuravleva et al., 2012). Despite common features, the mechanisms of activity are distinct in different families of chaperones ( Mattoo and Goloubinoff, 2014). Despite major advances in the field, how chaperones engage and alter the folding properties of non-native proteins remain poorly understood ( He et al., 2016 Huang et al., 2016 Koldewey et al., 2016 Libich et al., 2015 Rosenzweig et al., 2017 Saio et al., 2014 Sekhar et al., 2016 Verba et al., 2016 Wälti et al., 2017). Recent studies have also highlighted molecular chaperones as inhibitors of amyloid formation ( Mainz et al., 2015 Taylor et al., 2016). Thus, chaperones are central to protein homeostasis in the cell and are essential for life ( Hipp et al., 2014 Powers and Balch, 2013). Molecular chaperones typically prevent the aggregation and assist with the folding of non-native proteins ( Balchin et al., 2016 Bukau et al., 2006). Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood.
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