It is clear from this example of than the conformational selection mechanism, because of AdK that conformational ensembles of the free state pro- the common observation that the crystal structure of the vide a simple way to determine which mechanism is bound protein is different to that of the free molecule.
Despite the clarity of the Others have noted, and we stress here again, that a con- models above, the number of cases in which a given clusion of induced fit because of different snapshots of the mechanism has been clearly demonstrated is limited. In the free and bound proteins is not warranted Boehr et al. Similarly, the inability to detect low populated and discuss how they can be interpreted.
Recent advances enable the detection and characterisation of near- invisible, low populated stable states in the native ensem- Biomolecular recognition from ensembles ble Korzhnev et al. It will be seen below that structural heterogeneity can sometimes account for the The first examples of conformational selection mechanisms observed differences without the need to invoke an induced are starting to appear in the literature.
MD simulations fit mechanism. Discrimination of recognition mechanisms were used by Salsas-Escat and Stultz to generate purely on the basis of static structures is not possible and ensembles of type III collagen that describe its conforma- the use of methods that provide information on structural tional heterogeneity.
They observed that collagen could heterogeneity can indeed be of great use. Collagen contains additional cleavage sites for the proteolytic enzyme CMMP8 but these sites do not sample the bound configuration. These experi- ments suggest that proteolytic degradation of collagen is controlled by the formation of the correct collagen conformation, and the results are consistent with the experimentally observed cleavage pattern Fields Conformational selection has also been shown to be a viable molecular recognition model for nucleic acids.
This RNA contains a bulge, which is a source of structural heterogeneity in the mole- cule. The motion of the bulge was analysed by use of RDCs with the help of MD simulations to generate a conforma- tional ensemble that captures the dynamic sub-states of the Fig. Confor- system. Analysis of the ensemble revealed that the internal mational selection for ubiquitin where the bound and unbound motion of TAR RNA was equivalent to the structural conformations share large overlap yellow and red and induced fit for TIS11d where there is very little overlap of the distributions green changes observed in X-ray structures of TAR RNA in and blue.
Data for ubiquitin adapted from Qin et al. Ubiquitin complexes with its ligands. Examples of induced fit are less abundant, although a The distributions of pairwise RMSD to the average bound disorder to order transition upon binding provides a good structure are scaled to have the same volume example of this mechanism.
This protein folds induced fit and conformational sampling, and concluded upon binding to its cognate RNA to regulate RNA degra- that the role of induced fit was only marginally lower than dation Qin et al. We recently generated a Hudson et al. This exam- result suggests that conformational selection is likely to ple, where the induced fit mechanism seems to dominate, play a larger role than induced fit in the molecular recogni- does not exclude the presence of the conformational tion of ubiquitin Fenwick et al.
As shown in Fig. Their innovate technique gives further evidence of key to determining the population of the bound confor- the role of conformational selection in the binding of ubiq- mation in the unbound state by using MD or ensemble MD uitin partners via a population redistribution i. We A combination of conformational selection and induced highlight the differences between this example of confor- fit has been observed in ubiquitin binding.
More than 40 mational selection with that of induced fit for TIS11d in structures of ubiquitin in complex with binding partners are Fig. It can be seen that in the case of conformational available and show that ubiquitin can adopt different con- selection the bound and free states have low and similar figurations upon binding.
Lange et al. This result showed that sampling explained by conformational selection. They generated the free state was sufficient to reach the configuration of the conformational ensembles from S2 and NOE data for two bound state in a conformational selection mechanism, with peptide proteases inhibitors SGCI and SGTI and found in only minor induced fit changes to the backbone and side both cases that the conformational ensemble of the free chain rotameric states.
Wlodarski and Zagrovic states contained configurations corresponding to the further studied ubiquitin using global multidimensional structures of the inhibitors bound to the proteases Gaspari scaling analysis to determine the relative weights of et al. The recognition because in many cases the solution structure of MWC model assumes that these two conformations are in the RNA is markedly different from that observed in the equilibrium and that the motion of each of the binding sites complexes with RNA-binding proteins Williamson Wright and the bound conformation at both binding sites is visited even co-workers show that a lock and key mechanism can in the absence of ligand.
Gunasekaran et al. A second report of lock and key for RNA ulation shift of the ensemble. The KNF model is instead has also become available in which one of the molecules is conceptually related to the induced fit mechanism of static and does not change its conformation upon binding. Moreover, mutations that freeze through the propagation of conformational changes across out motion of the related TAR RNA have been shown to the structure of the protein.
The conformational spread increase the binding affinity Stelzer et al. These schemes are shown in Fig. It is interesting to consider the implications of these models for the motion of allosteric biomolecules in the absence of their ligands.
The MWC model, which explic- Allostery and correlated motion itly considers the dynamics of the free state, invokes strong correlation of the motions at each binding site. The KNF In the above examples, we have ignored the role of allostery model, instead, only requires binding in the first site to in molecular recognition. Allostery is the process by which influence the affinity of the second binding site for its the affinity of a binding site for a ligand is affected by the ligand.
These observations indicate that careful analysis of binding of a second ligand in a different, distant, site. Simple contact models mechanism by which binding allostery operates for specific can characterize networks of this type for some biomole- systems to be determined. It can be seen from Fig.
In their opinion, all interactions tional ensembles, double mutant cycles are one of the can potentially have allosteric consequences, because of strongest experimental validations of allosteric channels the nature of population shift, and can result in highly and are routinely used in their investigation.
These types of complex and redundant networks. Using these ideas, they data can be useful in that they can determine if cooperative were able to rationalise the complexity of biomolecular channels and mechanisms are present. In some cases they interaction networks that operate in transcriptional regu- can indicate which residues are involved in such channels. This work has recently been Determination of the underlying mechanisms is more dif- reviewed Pan et al.
Istomin et al. The schemes represent the identity of the possible states are possible there is a strong correlation between the conformations of a tetrameric a and heterodimer b allosteric conformation of each subunit. In the KNF model, instead, ligand protein that are present in solution as the concentration of ligand is binding causes a local conformational change in the subunit, that increased from top to bottom. Ligand binding is represented as a influences the affinity of the other subunits for the ligand without the change in color in the subunit from white to black whereas need to invoke structural changes; as the conformational changes in conformational change is represented by a change in shape; the the various binding sites are not concerted this model requires a populations of the various conformations are not represented.
In the weaker correlation between the conformations of each subunit. The MWC model the species in solution do not change but their general scheme of Eigen, where a full permutation of the states is populations shift as a consequence of ligand binding; as only two considered, is also shown right protein G and observed that the backbone order parameters In Cartesian space, the standard analysis is com- varied less than would be expected if the residues fluctu- putation of the distance correlation matrix; although this ated independently, suggesting widespread concerted method is sensitive to large displacements, it does not motion of pairs of residues.
This claim is controversial and report on the nature of the hinges that give rise to the could not be substantiated by MD simulations of this and observed displacements. Thus, if one is interested in other proteins Lange et al. The choice of internal coordinates dihedral Biomolecular allostery from ensembles space is natural for proteins because most of their motion is due to fluctuations in dihedral angles.
Correlated motion Descriptions of allostery from ensembles can be indicative can be analysed in dihedral space and can be used to of correlated motion. Because of the challenges in directly identify hinge regions. Thorough analysis of correlated determining time-resolved coordinates from experiments, motion requires analysis in both reference frames, although detection of correlations between the motion of residues it is preferable to use internal coordinates when trying to distant in sequence has remained elusive.
As described understand mechanistic details and to elucidate allosteric above, conformational ensembles are most often used for channels Clore and Schwieters a; Showalter and this purpose. We compare these two MD, and enable protein motion to be characterized at methods in Fig. Once the ensembles are generated, there are different Analysis of MD simulations has become commonplace ways of determining whether they contain correlated in Cartesian space, with examples covering a large range of motion.
One of the most important considerations is the proteins. Dihydrofolate reductase has non-additive behav- frame of reference for extracting the motion. If Cartesian iour in double mutant cycles and MD simulations Agarwal coordinates are chosen, the first difficulty is choosing the et al.
The simulations predicted the propagation of motion between strands of the b-sheet, consistent with the b-lever motion Fenwick et al. The drug Hoechst binds to duplex DNA. Extended MD simulations of the and complex of the drug and DNA showed that binding allostery is primarily a conse- quence of the transfer of dynamic information rather than of structural change Harris et al.
Careful experi- mental investigations combined with modelling enabled the observation of correlated motion that biases transitions along predetermined conformational pathways Fig. In this case the pathways of motion could be directly deter- mined from the experimental data, and by selecting frames from ensembles determined using unbiased MD it was possible to visualise correlated motion.
It seems that the Fig. Furthermore, it was possible to state. Following binding the ensemble undergoes a population shift, redistributing the conformational states. Both conformational selection and induced fit appear to play roles.
Following binding by a primary conformational selection event, optimization of side chain and backbone interactions is likely to proceed by an induced fit mechanism. These data support a new molecular recognition paradigm for processes as diverse as signaling, catalysis, gene regulation and protein aggregation in disease, which has the potential to significantly impact our views and strategies in drug design, biomolecular engineering and molecular evolution.
The role of dynamic conformational ensembles in biomolecular recognition. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. N2 - Molecular recognition is central to all biological processes.
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