Direct Visualization Reveals Dynamics of a Transient Intermediate During Protein Assembly

Abstract

Interactions between proteins underlie numerous biological functions. Theoretical work suggests that protein interactions initiate with formation of transient intermediates that subsequently relax to specific, stable complexes. However, the nature and roles of these transient intermediates have remained elusive. Here, we characterized the global structure, dynamics, and stability of a transient, on-pathway intermediate during complex assembly between the Signal Recognition Particle (SRP) and its receptor. We show that this intermediate has overlapping but distinct interaction interfaces from that of the final complex, and it is stabilized by longrange electrostatic interactions. A wide distribution of conformations is explored by the intermediate; this distribution becomes more restricted in the final complex and is further regulated by the cargo of SRP. These results suggest a funnel-shaped energy landscape for protein interactions, and they provide a framework for understanding the role of transient intermediates in protein assembly and biological regulation.EPR spectroscopy | fluorescence spectroscopy | molecular recognition | protein targeting | GTPases I nteractions between proteins are central to biology and underlie numerous molecular recognition, regulation, and signaling events (1). A challenge in our understanding of protein interactions is to reconcile their fast association kinetics required for biological function (10 6 -10 8 M −1 s −1 ) with the fact that formation of stable protein assemblies often involves extensive short-range, stereospecific interactions that are difficult to accomplish during a single diffusional encounter (2-4). This problem becomes more pronounced in protein interactions that require extensive conformational changes in the interaction partners. Much theoretical work has suggested that assembly of a protein complex initiates with the formation of a transient intermediate held together by solvent cage and long-range electrostatic attractions, followed by relative rotatory diffusions of the binding partners to search for the optimal interaction interface with shape and electrostatic complementarity (4-9). An extreme example of this concept is the "fly-casting mechanism," in which unstructured protein molecules bind targets weakly at a relatively large distance followed by folding at the target site (10-12). In general, formation of transient intermediates reduces the dimension of translational and rotational search and could significantly accelerate protein association.Despite significant progress in theoretical work, direct experimental demonstration of this model has been limited, and the structural and dynamic nature of transient intermediates during protein interactions has remained elusive. Experimental studies of transient intermediates are still at the infant stage, because these intermediates have short lifetimes and are rarely populated at equilibrium. Pioneering NMR studies have revealed the structures of rare conformational states in equilibrium with the predominant ...

Electrostatic Interactions in Protein Structure, Folding, Binding, and Condensation

“…542 Significant charge complementarity is present in the first three protein pairs. For FtsY and Ffh, charge complementarity is present to some degree between the N subdomains (toward the left), which were implicated in forming the interface of a kinetic intermediate, 543,544 but absent between the G subdomains (to the right), which form the interface in the native complex. 545,546…”

Receptor compaction and GTPase rearrangement drive SRP-mediated cotranslational protein translocation into the ER

“…The two NG domains undergo cooperative rearrangements upon their assembly, which culminate in the reciprocal activation of the GTPase activity of one another (4,5). The essential 4.5S RNA accelerates SRP-SR assembly 10 3 -fold to activate protein targeting, via a conserved GNRA tetraloop that makes transient contacts with the SR NG domain before the formation of the stable NG heterodimer (6)(7)(8). Compared to the bacterial homologs, eukaryotic SRP and SR underwent an extensive expansion in size and complexity.…”

“…1A) (14). The SR MoRF specifically accelerates SRP•SR assembly in response to the ribosome and was proposed to functionally replace the role of the 4.5S SRP RNA in bacterial SRP (7,8,15), but how it carries out these functions is unclear.…”

Activated GTPase movement on an RNA scaffold drives cotranslational protein targeting

“…SRP and FtsY undergo an unusual GTPase cycle, driven by multiple conformational rearrangements in their heterodimer that culminate in GTPase activation (Figs. 3a-d, cartoon) [22][23][24] . We asked how these rearrangements within the GTPase complex drive its global movements on the SRP RNA, using conditions that block the GTPase cycle at distinct stages 22,23 .…”

“…7a). Recruitment of FtsY begins with a transient early intermediate, which lacks close contacts between the G-domains and hence can be isolated by leaving out GTP analogues 12,24 . No GTPase movement was observed at this stage either (Fig.…”