A litmus test for classifying recognition mechanisms of transiently binding proteins

Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin’s paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks

A litmus test for classifying recognition mechanisms of transiently binding proteins

Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin’s paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks

NMR Derived Model of GTPase Effector Domain (GED) Self Association: Relevance to Dynamin Assembly

Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its ‘coiled coil’ GTPase Effector Domain (GED), which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the self-association of GED by first dissociating the assembly using Dimethyl Sulfoxide (DMSO) and then systematically monitoring the refolding into helix and concomitant re-association using NMR spectroscopy, as DMSO concentration is progressively reduced. The short segment, Arg109 - Met116, acts as the nucleation site for helix formation and self-association. Hydrophobic and complementary charge interactions on the surfaces drive self-association, as the helices elongate in both the directions resulting in an antiparallel stack. A small N-terminal segment remains floppy in the assembly. Following these and other published results on inter-domain interactions, we have proposed a plausible mode of dynamin self assembly

Detecting the Undetectable: Functional Protein Motions in the Hidden Timescale Window Revealed by NMR Relaxation Measurements

About 50 years ago, even before NMR was ready to determine structure of proteins, it could unveil a surprising motion in aromatic side-chain (Wuthrich & Wagner, 1975) from 1D 1H spectra. With decades of development, NMR is now the most powerful technique for studying atomic resolution dynamics in proteins at biological conditions, even inside live cells. Although NMR offers relaxation-based methods to determine motion on the entire dynamics timescale spectrum, accurate characterization of the supra-τc dynamics (4 ns-40 µs) was not possible due to technical limitations. Dynamics from this timescale window is indicated to play a decisive role in molecular recognition and binding events. By combining the advancement in hardware with smart design of pulse programs, we have developed a high-power RD (relaxation dispersion) method, which can accurately detect dynamics in this previously inaccessible window. By applying this newly developed high-power R1ρ RD, a hidden supra-τc motion was found in the first loop of the well-known protein GB3. From the timescale of motion measured at several supercooled temperatures, the activation energy for the loop-motion was estimated to be 65.6 kJ mol-1. Arrhenius extrapolation showed that the loop moves with a timescale of ~400 ns at physiological temperature of 308 K. Analyzing the 640-membered ERMD (Ensemble-Restrained-Molecular-Dynamics) RDC ensemble, we found elevated dynamics from higher fluctuation of backbone atoms as well as lower supra-τc order parameters in the region where RD is detected. Interestingly, the newly observed supra-τc motion takes place in a region, which binds to antibodies. This hints to a link of the observed motion with the antibody recognition of GB3. After unveiling a functional backbone motion in GB3, in chapter 2 we have studied the dynamics of methyl groups in the side-chains of ubiquitin, with a new type of RD method; 13C Extreme-CPMG (E-CPMG), developed in our group. This method can cover the detectable timescale range of both conventional CPMG and R1ρ. E-CPMG reported the same timescale and amplitude of motion, which was previously found from R1ρ measurements with much longer measurements time. Similarity in timescale and activation energy of the detected side-chain motion with previously found backbone dynamics hints towards a common mechanism. This hypothesis was proven by the absence of the side-chain motion in two different single point mutants (E24A and G53A), which were designed and tested for the quenching of backbone dynamics. In chapter 3, we have extended the E-CPMG approach to a nucleus (1H) with higher gyromagnetic ratio, where we could generate higher B1 field with less applied RF power. We found and corrected a linear decay in RD profiles, arising from the phase cycle, which is widely used even in conventional CPMG measurements. Using this approach, we could detect the peptide-flip induced breathing motion in twice as many residues in ubiquitin compared to a previous report. In addition, we could directly detect the large amplitude pincer-mode motion for the first time, in segments where the existence of supra-τc motion was predicted from both RDC and MD simulations. This newly detected motion was already predicted to contribute to the conformational adaption power of ubiquitin while binding to other proteins. Finally, in chapter 4, we found a very fast (4 µs) dynamics at 263 K in an intrinsically disordered protein p53-TAD, in residues which are found to be in helical conformation in a bound complex. So far, this is the fastest detected motion with RD. The helical propensity was also predicted in the same stretch of residues from SSP (secondary structural propensity) score calculation with the experimental chemical shifts of the free protein. A great reduction in exchange rate (~100 times) was found when the proline residue at the C-terminal of the helix was removed. In addition to that, more residues, including some from a second helix, were found to undergo conformational exchange. A doubling in helical propensity was found in this mutant (P27A) from SSP score calculations. In two other mutants (W23A; F19A and W23A; F19A; P27A) where the hydrophobic residues with aromatic side-chains were removed, no conformational exchange was detected. These finding suggests that the RD-observed 4 µs motion could originate from fast folding of the transiently formed helix which is assisted by the hydrophobic core in the center. In this thesis, by applying high-power RD, functionally relevant supra-τc motion is discovered in both backbone and side-chains of well-folded and disordered proteins. These findings helped in understanding molecular recognition and folding processes in the studied proteins.2022-05-2

Dynamin tubes around the neck of invaginating vesicles.

<p>Schematic representation of the organization of dynamin assembly wrapped around the neck of invaginating vesicles of parent membrane. The anti-parallel organization of the GED units results in a zigzag orientation of dynamin units which favor efficient binding of the dynamin tube around the neck of the vesicles. The PH domain binds to the lipid vesicles forming the base followed by GED and Middle domain forming a neck from which the GTPase domain protrudes as a head.</p

Summary of the gradual disappearance of the peaks with decreasing DMSO-d6 concentrations.

<p>(<b>A</b>) ¹H-¹⁵N HSQC spectra of GED in 100%, 90%, 85%, 80%, 70% and 50% DMSO-d6 at 45°C, as marked in the left hand panel within the spectra. (<b>B</b>) The additional residues disappearing in the HSQC spectra with decreasing DMSO concentrations are color coded on the primary sequence of GED as: 100% (olive), 90% (cyan), 85% (pink), 80% (red), 70% (green) and 50% (orange).</p

Models of dynamin assembly.

<p>(<b>A</b>) Schematic representation of the T-shaped dynamin tetramers or ‘dimer of dimers’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030109#pone.0030109-Blackstone1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030109#pone.0030109-Schmid1" target="_blank">[27]</a> which explains the inter-domain interactions between the GED, middle Domain and GTPase domain. (<b>B</b>) A hypothetical model derived from the present observations for formation of an elongated oligomer of dynamin. The 4 cylinders of different colors indicate 4 different domains of dynamin: GTPase domain, Middle domain, GED and Proline – Arginine rich domain. Pleckstrin Homology domain is indicated by a twisted sheet. The arrangement of the dynamin monomers is such that the GED domains are antiparallel to each other. The reported interactions of GTPase domain and Middle domain with GED and self interaction of GED are denoted by double headed arrows (solid line for intermolecular interactions whereas dashed line for intramolecular interactions). The entire assembly is fitted inside a box to provide a 3D view of the molecules. The extended dashed lines in different direction indicate that the assembly can extend both vertically and horizontally. (<b>C</b>) Representation of intramolecular back-folding of the GED domain onto the GTP-middle domain as suggested by Zhu et al (2004) which is well in accordance with our model.</p

A possible model for concomitant folding of the GED chain and association by DMSO dilution.

<p>(<b>A</b>) The polar amino acid rich segment of the completely or partially dissociated polypeptide chain of GED monomer have high probability of helix formation due to the electrostatic interaction. The monomeric units come in close proximity of other similar units in the system due to intermolecular charge interactions thereby forming clusters. (<b>B</b>) Helix formation initiates in a certain segment of the C-terminal of the monomer and the nascent helices of the monomers stack together to form barrel like structures. (<b>C</b>) The helix extends in the neighboring segments. The vacuum electrostatic potential surfaces generated by NOC software (<a href="http://noch.sourceforge.net/" target="_blank">http://noch.sourceforge.net/</a>) depict predominance of opposite charges in the segments Lys68 – Glu80 and Asp95 – Lys121, which favors anti-parallel organization of the newly formed helices of the monomeric units to form extended oligomeric units. (<b>D</b>) Helix formation initiates in the N-terminal half of the monomer. However Proline 67 acts as a kink around which the polypeptide chain can bend thereby providing certain degree of flexibility to the N-terminal half. These units simultaneously arrange themselves in a particular orientation such that the N-terminal helices of the monomeric units are not completely embedded in the core of the assembly; rather it has certain degree of flexibility. (<b>E</b>) The N-terminal helix is completely formed and the oligomers arrange themselves linearly in random numbers to form the extended oligomers with a wide diversity of size. (<b>F</b>) Enlarged view of a part of the TEM image of copper plate with ∼1 µM GED in water (inset) at room temperature. The image shows differential size and shape distribution of GED oligomers, although the majority show elongated structures. The concentration of the protein mentioned is not the true concentration due to the evaporation of water during sample preparation.</p

Structural propensities of the equilibrium states of GED with DMSO–d6 dilution.

<p>(<b>A</b>) Residue-wise plots of C^α secondary chemical shifts of GED in 100%, 90%, 85% and 80% DMSO-d6 at 45°C. Positive deviations of C^α indicate alpha helical propensities whereas negative deviations indicate beta preferences. The deviations of C^α chemical shifts at different DMSO concentrations were measured by using the formula, ΔC^α = C_obs−(p1*C_ran,dmso+p2*C_ran,aq) ,where p1 and p2 are the relative contributions of random coil chemical shifts in DMSO and aqueous environment, respectively. The straight line in the plots is drawn at 0.7 ppm to indicate the uncertainty in the spread of C^α chemical shifts. (<b>B</b>) Residue wise plots of the secondary ³JH^N_-H^α coupling constants of GED in 100%, 90% and 85% DMSO-d6. At 85% DMSO, the splittings of only the N-terminal residues could be measured. The high-resolution HSQCs of GED in lower concentrations of DMSO did not show any measurable splitting. The empty and the filled black bars at the top of the graphs represent the helical and extended beta sheet like propensities.</p

Slow conformational exchange in the equilibrium states.

<p>Illustrative representation of the residues showing two sets of peaks in the HSQC spectra at various DMSO dilutions, as indicated at the top left corner of the spectrum. The alternate sets of peaks in the HSQC spectra for the residues are indicated by primed and unprimed annotations. The boxed primed residues in the HSQC spectra indicate the similar positions of the alternate sets of peaks at various DMSO concentrations.</p