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 ...

Conformational properties of cardiolipin-bound cytochrome c

“…Data analysis of the TR-FRET was done in MATLAB (MathWorks) as previously described (16,47); for specific details see SI Materials and Methods. Critical distance R 0 calculations and analyses of the orientation factor κ 2 effects on the D-A distances are provided in SI Materials and Methods.…”

Translation Elongation Regulates Substrate Selection by the Signal Recognition Particle

“…We used a series of signal sequence variants described previously (37)(38)(39), in which the hydrophobic core of the pPL signal sequence was replaced by that from phoA, a borderline SRP substrate, or by a combination of leucine and alanine ( Table 1). The Leu/Ala ratio was varied to generate signal sequences with different hydrophobicity (Table 1).…”

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

“…1A). Both SRP and SRP receptor (called FtsY in bacteria) also contain a conserved NG domain, comprised of a GTPase (guanosine 5′-triphosphate hydrolase) G domain and the N domain, whose direct interaction mediates the delivery of cargo to the target membrane.Biophysical analyses (32)(33)(34) showed that membrane targeting is a two-step process in which SRP and FtsY first associate via their N domains to form a transient early intermediate (31,32,35). GTP (guanosine 5′-triphosphate)-driven rearrangements then bring the G domains of both proteins into close contact, giving a stable closed complex (36,37).…”