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.

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 Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation.

Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin.

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.

Site-specific fluorescence dynamics in an RNA 'thermometer' reveals the role of ribosome binding in its temperature-sensitive switch function.

RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20-45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine-Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA.

Liaison between myristoylation and cryptic EF-hand motif confers Ca(2+) sensitivity to neuronal calcium sensor-1.

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kß). Of a particular note, Ca(2+) induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca(2+)-binding to EF-1 is restored. Moreover, Ca(2+), which otherwise augments the enzymatic activity of PI4Kß (modulated by NCS-1), leads to a further decline in the modulated PI4Kß activity by myristoylated mutants (with canonical EF-1) pointing toward a loss of Ca(2+) signaling and specificity at the structural as well as functional levels. This study establishes the presence of the strong liaison between myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of Ca(2+) specific signal transduction to downstream effecter molecules despite Ca(2+) binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca(2+) sensitivity/specificity in NCS-1.

Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin.

Conformational propensities and dynamics of a ßγ-crystallin, an intrinsically disordered protein.

The three-dimensional folded structure of a protein has been considered essential for its function. However, recently many proteins have been identified to function without having a definite structure and they have been classified as intrinsically disordered proteins (IDPs). Recently, we have identified a ßγ-crystallin domain in the genome of a marine bacterium called Hahella chejuensis on the basis of known sequence signatures. This protein, called Hahellin, was characterized by NMR spectroscopy as an IDP, which upon Ca(2+)-binding was shown to undergo a large conformational transformation and acquires a typical ßγ-crystallin fold. In this paper, we have characterized this IDP by a combined use of NMR and Replica Exchange Molecular Dynamics simulation and found it to be in a highly dynamic, inter-converting population having a molten globular state with the C-terminal Greek key motif relatively more flexible as compared to its N-terminal counterpart. Network analysis and clustering on the observed conformational ensemble showed a heterogeneous mixture of eleven distinct clusters, classified into near-native and far-native populations, which are not in equilibrium. Several conformational clusters display an increased propensity for helical conformation(s) and a decreased ß-strand propensity, which is consistent with the NMR observations made on this protein. The negatively charged Ca(2+)-coordinating residues form parts of the highly flexible polypeptide stretches, and thus act as seeds for the origin of different conformational clusters observed. This study thus helps us to understand the relationship between the role of conformational dynamics and the structural propensities of the intrinsically disordered state of apo-Hahellin.

Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the ßγ-crystallin superfamily from Methanosarcina acetivorans.

ßγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two ß-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens ßγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens ßγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.

Calmodulin-like protein from Entamoeba histolytica: solution structure and calcium-binding properties of a partially folded protein.

The mechanism of Ca(2+)-signaling in the protozoan parasite Entamoeba histolytica is yet to be understood as many of the key regulators are still to be identified. E. histolytica encodes a number of multi-EF-hand Ca(2+)-binding proteins (EhCaBPs). Functionally only one of these molecules, EhCaBP1, has been characterized to date. The calmodulin-like protein from E. histolytica (abbreviated as EhCaM or EhCaBP3) is a 17.23 kDa monomeric protein that shows maximum sequence identity with heterologous calmodulins (CaMs). Though CaM activity has been biochemically shown in E. histolytica, there are no reports on the presence of a typical CaM. In an attempt to understand the structural and functional similarity of EhCaM with CaM, we have determined the three-dimensional (3D) solution structure of EhCaM using NMR. The EhCaM has a well-folded N-terminal domain and an unstructured C-terminal counterpart. Further, it sequentially binds only two calcium ions, an unusual mode of Ca(2+)-binding among the known CaBPs, notably both in the N-terminal domain of EhCaM. Further, EhCaM is present in the nucleus in addition to the cytoplasm as detected by immunofluorescence staining, unlike other EhCaBPs that are detected only in the cytoplasm. Therefore, this protein is likely to have a different function. The presence of unusual and a diverse set of CaBPs in E. histolytica suggests a distinct Ca(2+)-signaling process in E. histolytica. The results reported here help in understanding the structure-function relationship of CaBPs including their Ca(2+)-binding properties.

Temperature-dependent oligomerization in M-crystallin: lead or lag toward cataract, an NMR perspective.

The oligomerization and/or aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from abnormal disease states like Alzheimer's and Parkinson's disease to the production of inclusion bodies, stability, and delivery of protein drugs. In the case of eye-lens proteins, oligomerization is implicated in cataract formation. In the present study, we have investigated the temperature driven oligomerization of M-crystallin, a close homologue of eye-lens proteins, using NMR spectroscopy and dynamic-light scattering (DLS). The NMR data primarily included R(1), R(2) relaxation rates and nOes of the backbone amide groups recorded at three different temperatures, 25, 20, and 15° C. The major outcome of the study is the two fold increase in the overall tumbling time (τ(c)) of M-crystallin on lowering the temperature from 25 to 15° C. An extrapolation of τ(c) to a further lower temperature (5° C) may lead to a τ(c) of ∼19 ns that would correspond to a τ(c) value of a tetrameric M-crystallin. These results also validate the observed changes in the hydrodynamic radius of M-crystallin, determined using DLS data. Further, the temperature-dependent protein dynamics of M-crystallin reveal considerable variation at/near the Ca(2+)-binding sites. A concerted analysis of the temperature dependent relaxation parameters and DLS data reveals that the self-association of the protein is not only a monomer-dimer equilibrium, but also goes to tetramers or other multimeric states. These higher states may co-exist in fast exchange with the monomeric and dimeric M-crystallin at milli-molar to sub-millimolar concentrations and at lower temperature.

Biophysical Reviews' "Meet the Councilor Series"-a profile of Kandala V. R. Chary.

It gives me great pleasure to introduce myself to the readers of Biophysical Reviews. I share a brief account of my career and experiences in biophysical research spanning four decades. For the most of this period, I have worked at the Tata Institute of Fundamental Research (TIFR), Mumbai and Hyderabad.

A natively unfolded ßγ-crystallin domain from Hahella chejuensis.

To date, very few ßγ-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative ßγ-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a ßγ-crystallin fold is not known. We describe here a ßγ-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other ßγ-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical ßγ-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial ßγ-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded ßγ-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily.

Root-mean-square-deviation-based rapid backbone resonance assignments in proteins.

We have shown that the methodology based on the estimation of root-mean-square deviation (RMSD) between two sets of chemical shifts is very useful to rapidly assign the spectral signatures of (1)H(N), (13)C(α), (13)C(ß), (13)C', (1)H(α) and (15)N spins of a given protein in one state from the knowledge of its resonance assignments in a different state, without resorting to routine established procedures (manual and automated). We demonstrate the utility of this methodology to rapidly assign the 3D spectra of a metal-binding protein in its holo-state from the knowledge of its assignments in apo-state, the spectra of a protein in its paramagnetic state from the knowledge of its assignments in diamagnetic state and, finally, the spectra of a mutant protein from the knowledge of the chemical shifts of the corresponding wild-type protein. The underlying assumption of this methodology is that, it is impossible for any two amino acid residues in a given protein to have all the six chemical shifts degenerate and that the protein under consideration does not undergo large conformational changes in going from one conformational state to another. The methodology has been tested using experimental data on three proteins, M-crystallin (8.5 kDa, predominantly ß-sheet, for apo- to holo-state), Calbindin (7.5 kDa, predominantly α-helical, for diamagnetic to paramagnetic state and apo to holo) and EhCaBP1 (14.3 kDa, α-helical, the wild-type protein with one of its mutant). In all the cases, the extent of assignment is found to be greater than 85%.

Statistical analysis of intermolecular interactions in trypsin-inhibitor complexes.

Communicated by Ramaswamy H. Sarma.

NMR structure and dynamics of inhibitory repeat domain variant 12, a plant protease inhibitor from Capsicum annuum, and its structural relationship to other plant protease inhibitors.

Although several plant protease inhibitors have been structurally characterized using X-ray crystallography, very few have been studied using NMR techniques. Here, we report an NMR study of the solution structure and dynamics of an inhibitory repeat domain (IRD) variant 12 from the wound-inducible Pin-II type proteinase inhibitor from Capsicum annuum. IRD variant 12 (IRD12) showed strong anti-metabolic activity against the Lepidopteran insect pest, Helicoverpa armigera. The NMR-derived three-dimensional structure of IRD12 reveals a three-stranded anti-parallel ß-sheet rigidly held together by four disulfide bridges and shows structural homology with known IRDs. It is interesting to note that the IRD12 structure containing ∼75% unstructured part still shows substantial amount of rigidity of N-H bond vectors with respect to its molecular motion.Communicated by Ramaswamy H. Sarma.