Fundamental Challenges and Outlook in Simulating Liquid-Liquid Phase Separation of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) populate an ensemble of dynamic conformations, making their structural characterization by experiments challenging. Many IDPs undergo liquid-liquid phase separation into dense membraneless organelles with myriad cellular functions. Multivalent interactions in low-complexity IDPs promote the formation of these subcellular coacervates. While solution NMR, Förster resonance energy transfer (FRET), and small-angle X-ray scattering (SAXS) studies on IDPs have their own challenges, recent computational methods draw a rational trade-off to characterize the driving forces underlying phase separation. In this Perspective, we critically evaluate the scope of approximation-free field theoretic simulations, well-tempered ensemble methods, enhanced sampling techniques, coarse-grained force fields, and slab simulation approaches to offer an improved understanding of phase separation. A synergy between simulation length scale and model resolution would reduce the existing caveats and enable theories of polymer physics to elucidate finer details of liquid-liquid phase separation (LLPS). These computational advances offer promise for rigorous characterization of the IDP proteome and designing peptides with tunable material and self-assembly properties.

The structural biology of crystallin aggregation: challenges and outlook.

Crystallin aggregation is characterized by light scattering of large molecular aggregates due to their phase separation in the lens. Low-resolution biophysical studies using multiple techniques have characterized the folding, stability, binding, and aggregation of crystallins in the past but with limited access to their structure, dynamics, and interactions. In this Viewpoint, three schools of experimental structural biology, that is, X-ray crystallography, solution and solid-state NMR spectroscopy, and cryo-electron microscopy, combine to provide atomic resolution details of native crystallins, soluble oligomers, and insoluble amyloid fibrils and amorphous aggregates. Computational structural biology provides additional details on crystallin dynamics and the crucial intercrystallin interactions in these events. Our current understanding of the diverse structural biology of crystallins is consistent with multiple pathways of protein aggregation for different structural intermediates. This Viewpoint combines our efforts with those of others to elucidate the recent progress in these high-resolution studies and proposes an integrated structural biology approach to resolve the complex lens interactome. Overall, I discuss the outstanding questions and evaluate the experimental and theoretical caveats in the field.

A Perspective on Biophysical Studies of Crystallin Aggregation and Implications for Cataract Formation.

Lens crystallins are subject to various types of damage during their lifetime which triggers protein misfolding and aggregation, ultimately causing cataracts. There are several models for crystallin aggregation, but a comprehensive picture of the mechanism of cataract is still underway. The complex biomolecular interactions underlying crystallin aggregation have motivated major efforts to resolve the structural details and mechanism of aggregation using multiple biophysical techniques at different resolutions. Together, experimental and computational approaches identify and characterize both amyloidogenic and amorphous aggregates leading to an improved understanding of crystallin aggregation. A rigorous characterization of the aggregation-prone intermediates is crucial in cataract-mediated drug discovery. This Perspective summarizes recent biophysical studies on lens crystallin aggregation. We evaluate the outstanding challenges, future outlook, and rewards in this fertile field of research. With lessons learned from protein folding and multiple pathways of aggregation, we highlight the differences in the overall mechanisms of age-related and congenital cataracts. We expect that a correlation between the existing and developing biophysical techniques would provide a platform to study amyloid architecture in the eye lens and reduce the existing gaps in our understanding of crystallin biophysics.

A Molecular Dynamics Perspective To Identify Precursors to Aggregation in Human γS-Crystallin Unravels the Mechanism of Childhood Cataracts.

Despite the increasing health risk from infantile cataracts, identifying the mechanism of this disease remains a challenge due to a lack of structural investigations using experimental and computational approaches. Mutations in human γS-crystallin are contingent with childhood cataracts. Our recent high-resolution structural studies using solution NMR spectroscopy established the key role of the G57W mutation in human γS-crystallin (abbreviated hereafter as γS-G57W), promoting structural instability. In order to design therapeutics to delay or upset congenital cataracts, the characterization of the precursors to γS-G57W aggregation is indispensable. In this endeavor, we present microsecond long unbiased atomistic molecular dynamics simulations and principal component analyses that unfold insights into lens crystallin aggregation. An enhanced sampling metadynamics approach was further employed to systematically unravel the molecular dynamics underlying crucial interdomain contacts. Taken together, our experiment-guided computational study in this paper led to the identification of domain-swapped intermediates in γS-G57W to atomic resolution with insights into the aggregation of lens crystallins causing childhood cataracts for the first time with functional consequences.

Structural studies on the individual domains of human γS-crystallin and its G57W mutant unfolds mechanistic insights into childhood cataracts.

Inter-domain interactions tune the exceptional stability of human γS-crystallin (γS-WT) in the eye lens, which lasts a lifetime with no protein turnover. Our recent NMR studies revealed the key role of G57W mutation in γS-WT, as the familial determinate of childhood cataracts. As the unusually exposed W57 is near the inter-domain interface, a recurring theme of this study is the upsetting of inter-domain contacts exposing hydrophobic patches, which may initiate aggregation at crystallin concentrations not so surprising in the eye lens. In this endeavour, to untangle the mechanistic pathways triggering aggregation in the cataract variant γS-G57W, we undertook high-resolution structural characterization of isolated domains vis-a-vis full length γS-crystallin. Here we report for the first time, thermodynamic and kinetic determinants of structural stability with their eccentric shifts under pathological stress employing sophisticated spectroscopy techniques. We propose that domain interface acts as an intrinsic stabilizer for the otherwise floppy N-terminal domain in γS-G57W than in γS-WT where it serves an extrinsic role. Our results present a residue resolved quantitative analysis for differential domain stabilities from non-linear temperature coefficients of 1HN chemical shifts using solution NMR spectroscopy. Consistent with the Ca2+-binding episode that lasted poorly for human lens crystallins, our results show that disease mutants attenuate it further and completely silence it in extreme cases. Overall, our study provides a compelling evidence for the diverse structural evolution of lens crystallins elucidating molecular details to apprehend lens opacification and suggests the scope of therapeutics in reducing the global trauma of cataracts.

On identifying low energy conformational excited states with differential ruggedness in human γS-crystallin promoting severe infantile cataracts.

Transient excited states in proteins can be accurately probed from temperature dependence of amide proton (1HN) chemical shifts displaying significant curvatures. Characterizing these near-native alternative states is of high therapeutic relevance in conformational diseases wherein missense mutations promote structural instability that leads to conformational heterogeneity. Extending the structure-function paradigm from physiology to pathology, we recently reported the solution NMR structure and dynamics of a severe congenital cataract variant, G57W of human γS-crystallin (abbreviated as γS-G57W) which is resistant towards crystallization. In an endeavour to explore the functional consequences of this mutation, here we report for the first time, native state conformational ruggedness in human γS-G57W as compared to its wild-type counterpart from residue resolved nonlinear temperature dependence of backbone 1HN chemical shifts using solution NMR spectroscopy. Our calculations suggest that the simulated chemical shift curvatures are indicative of low energy excited states within 2-4 kcal mol-1 from the native state. Residues accessing alternative conformations populate the N-terminal domain of γS-G57W more than its C-terminal counterpart. Collectively, curvatures retaining native state ensemble on mild denaturation suggest that the free energy landscape in human γS-G57W at the bottom of the folding funnel is sufficiently robust and malleable against such perturbations. Overall, this critical study highlights the functional aspects of such structural malleability promoting infantile cataracts as a global health risk marker.

Enhanced H/D exchange unravels sequential structural excursions in G57W variant of human γS-crystallin with pro-cataractogenic conformations.

Our two recent reports on the high resolution NMR structure and conformational dynamics of G57W variant of human γS-crystallin (abbreviated as γS-G57W) causing severe infantile cataracts, revealed slackening of its N-terminal domain with enhanced local conformational dynamics attributed to mutation. Exploring the biochemistry of infantile cataracts in detail, here we studied structural unfolding in both human γS-WT and γS-G57W at residue level resolution using solution NMR spectroscopy and chemical kinetics and characterized the molecular intermediates with functional consequences. We report, for the first time, that human γS-crystallin unfolds sequentially under H/D exchange. This communication forms the first experimental evidence for non-concerted destabilization of structural foldon units in human γS-G57W. Residues contributing to the compact fold and structural stability exchanged their amide protons with deuterons more readily in γS-G57W compared to γS-WT, displaying differential free energies of exchange. Overall, our results establish a direct conformational link between the structure, dynamics, design and function in human γS-crystallin such that the G57W cataract variant promotes enhanced structural excursions concomitant with increased instability, elucidating very crucial molecular details of cataract formation affecting infants across the globe.

Conformational dynamics study on human γS-crystallin as an efficient route to childhood blindness.

Single point mutants of human γS-crystallin cause dominant congenital cataracts, a recent one of which involves the substitution of highly conserved glycine at 57th position with a bulkier tryptophan. Our high-resolution 3D structure of this G57W mutant (abbreviated hereafter as γS-G57W), reported recently revealed site-specific structural perturbations with higher aggregation and lower stability compared to its wild-type; a structural feature associated with important functional and therapeutic consequences. In this communication, we report for the first time, residue resolved conformational dynamics in both γS-WT and γS-G57W using solution NMR spectroscopy, and suggest how these differences could crucially affect the biochemistry of the mutant. Guided by our critical structural investigations, extensive conformational dynamics and biophysical studies presented here show that loss of structural stability arises from enhanced dynamics in Greek key motif 2 inducing flexibility in the N-terminal domain as opposed to its structurally unperturbed C-terminal counterpart. NMR spectral density correlations and internal dynamics comparisons with the wild-type suggest that the overall thermodynamic instability propagates from the mutated N-terminal ß4-ß5 loop providing a residue level understanding of the structural changes associated with this early onset of lens opacification. Our results highlight the vital role of conserved Greek key motifs in conferring structural stability to crystallins and provide crucial molecular insights into crystallin aggregation in the eye lens, which triggers cataract formation in children. Overall, this critical study provides a residue level understanding of how conformational changes affect the structure and function of crystallins in particular and proteins in general, during health and disease.

Structure of G57W mutant of human γS-crystallin and its involvement in cataract formation.

A recently identified mutant of human γS-crystallin, G57W is associated with dominant congenital cataracts, the familial determinate of childhood blindness worldwide. To investigate the structural and functional changes that mediate the effect of this cataract-related mutant to compromise eye lens transparency and cause lens opacification in children, we recently reported complete sequence-specific resonance assignments of γS-G57W using a suite of heteronuclear NMR experiments. As a follow up, we have determined the 3D structure of γS-G57W and studied its conformational dynamics by solution NMR spectroscopy. Our structural dynamics results reveal greater flexibility of the N-terminal domain, which undergoes site-specific structural changes to accommodate W57, than its C-terminal counterpart. Our structural inferences that the unusual solvent exposure of W57 is associated with rearrangement of the N-terminal domain suggest an efficient pathway for increased aggregation in γS-G57W and illuminates the molecular dynamics underlying cataractogenic aggregation of lens crystallins in particular and aggregation of proteins in general.

Sequence specific 1H, 13C and 15N resonance assignments of the C-terminal domain of human γS-crystallin.

The high solubility and stability of crystallins present in the human eye lens maintains its transparency and refractive index with negligible protein turnover. Monomeric γ-crystallins and oligomeric ß-crystallins are made up of highly homologous double Greek key domains. These domains are symmetric and possess higher stability as a result of the complex topology of individual Greek key motifs. γS-crystallin is one of the most abundant structural ßγ-crystallins present in the human eye lens. In order to understand the structural stability of individual domains of human γS-crystallin in isolation vis-à-vis full length protein, we set out to structurally characterize its C-terminal domain (abbreviated hereafter as γS-CTD) by solution NMR. In this direction, we have cloned, over-expressed, isolated and purified the γS-CTD. The 2D [15N-1H] HSQC recorded with uniformly 13C/15N labeled γS-CTD showed a highly dispersed spectrum indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-CTD using a suite of heteronuclear 3D NMR experiments.

Structural and functional characterization of a missense mutant of human γS-crystallin associated with dominant infantile cataracts.

Infantile cataracts constitute one of the most important causes of childhood blindness worldwide. Human γS-crystallin is the most abundant protein in the eye lens cortex. A missense mutant of human γS-crystallin, Y67N (abbreviated hereafter as γS-Y67N) is recently reported to be associated with dominant infantile cataracts. To understand the structural basis for γS-Y67N to cause lens opacification, we constructed, expressed and purified γS-Y67N and its wild-type (abbreviated hereafter as γS-WT) and studied the structural and functional differences between them in solution using circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy and extrinsic spectral probes. Extensive equilibrium characterization indicate that replacement of the highly conserved Tyr at 67th position by Asn distorts the conserved Tyr corner at the second Greek key motif in the N-terminal domain (NTD) and leads to substantial loss of structural stability. Our intrinsic fluorescence quenching results reveal differential in-vitro refolding kinetics identifying partially folded kinetic intermediates for both proteins. Extrinsic fluorescence studies further reveal loosening up of the compact structure of γS-crystallin upon mutation associated with enhanced aggregation. As Ca2+ homeostasis is a crucial regulator of lens transparency, we further investigated the Ca2+-binding properties of γS-WT and γS-Y67N by isothermal titration calorimetry (ITC) to identify lens Ca2+ distribution in health and in disease. Overall, our results highlight the vital role of conserved Tyr corners in stabilizing Greek key motifs and provide useful structural and functional insights into the mechanism of cataract formation in humans.

Sequence specific 1H, 13C and 15N resonance assignments of a cataract-related variant G57W of human γS-crystallin.

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [15N-1H]-HSQC spectrum recorded with uniformly 13C/15N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific 1H, 13C and 15N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.