NMR insights into ß-Lactamase activity of UVI31+ Protein from Chlamydomonas reinhardtii.

ß-Lactamases (EC 3.5.2.6) confer resistance against ß-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against ß-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative ß-lactamase activity, sulbactam binding (a ß-lactam analogue) in the low µM affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known ß-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its ß-lactamase activity. Current study is the first report on ß-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for ß-lactamase activity.

The First Chemically-Synthesised, Highly Immunogenic Anti-SARS-CoV-2 Peptides in DNA Genotyped Aotus Monkeys for Human Use.

Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped Aotus monkeys and matched with HLA-DRß1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world's population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides' potential coverage for the world populations up to 62.9%. These peptides' 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology's potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human population.

Enhancing NMR derived ensembles with kinetics on multiple timescales.

Nuclear magnetic resonance (NMR) has the unique advantage of elucidating the structure and dynamics of biomolecules in solution at physiological temperatures, where they are in constant movement on timescales from picoseconds to milliseconds. Such motions have been shown to be critical for enzyme catalysis, allosteric regulation, and molecular recognition. With NMR being particularly sensitive to these timescales, detailed information about the kinetics can be acquired. However, nearly all methods of NMR-based biomolecular structure determination neglect kinetics, which introduces a large approximation to the underlying physics, limiting both structural resolution and the ability to accurately determine molecular flexibility. Here we present the Kinetic Ensemble approach that uses a hierarchy of interconversion rates between a set of ensemble members to rigorously calculate Nuclear Overhauser Effect (NOE) intensities. It can be used to simultaneously refine both temporal and structural coordinates. By generalizing ideas from the extended model free approach, the method can analyze the amplitudes and kinetics of motions anywhere along the backbone or side chains. Furthermore, analysis of a large set of crystal structures suggests that NOE data contains a surprising amount of high-resolution information that is better modeled using our approach. The Kinetic Ensemble approach provides the means to unify numerous types of experiments under a single quantitative framework and more fully characterize and exploit kinetically distinct protein states. While we apply the approach here to the protein ubiquitin and cross validate it with previously derived datasets, the approach can be applied to any protein for which NOE data is available.

Structural characterization of a novel KH-domain containing plant chloroplast endonuclease.

Chlamydomonas reinhardtii is a single celled alga that undergoes apoptosis in response to UV-C irradiation. UVI31+, a novel UV-inducible DNA endonuclease in C. reinhardtii, which normally localizes near cell wall and pyrenoid regions, gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV-stress. Solution NMR structure of the first putative UV inducible endonuclease UVI31+ revealed an α1-ß1-ß2-α2-α3-ß3 fold similar to BolA and type II KH-domain ubiquitous protein families. Three α-helices of UVI31+ constitute one side of the protein surface, which are packed to the other side, made of three-stranded ß-sheet, with intervening hydrophobic residues. A twenty-three residues long polypeptide stretch (D54-H76) connecting ß1 and ß2 strands is found to be highly flexible. Interestingly, UVI31+ recognizes the DNA primarily through its ß-sheet. We propose that the catalytic triad residues involving Ser114, His95 and Thr116 facilitate DNA endonuclease activity of UVI31+. Further, decreased endonuclease activity of the S114A mutant is consistent with the direct participation of Ser114 in the catalysis. This study provides the first structural description of a plant chloroplast endonuclease that is regulated by UV-stress response.

The Structure of Melanoregulin Reveals a Role for Cholesterol Recognition in the Protein's Ability to Promote Dynein Function.

Melanoregulin (Mreg) is a small, highly charged, multiply palmitoylated protein present on the membrane of melanosomes. Mreg is implicated in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these organelles. The possible molecular function of Mreg was identified by solving its structure using nuclear magnetic resonance (NMR) spectroscopy. Mreg contains six α helices forming a fishhook-like fold in which positive and negative charges occupy opposite sides of the protein's surface and sandwich a putative, cholesterol recognition sequence (CRAC motif). Mreg containing a point mutation within its CRAC motif still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Moreover, wild-type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, reversing the charge of three clustered acidic residues partially inhibits Mreg's ability to drive these organelles to microtubule minus ends.

Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

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.

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.

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%.

Sequence specific 1H, 13C and 15N backbone resonance assignments of UVI31+ from Chlamydomonas reinhardtii.

The cDNA of UVI31+ was cloned from C. reinhardtii and expressed in E. coli from where the protein was purified to homogeneity. The purified protein exhibited beta-lactamase activity (Manuscript in preparation). However, UVI31+ has no homology with the known ß-lactamases. In order to understand the structural basis of the ability of UVI31+ to hydrolyze ß-lactam antibiotics, we in parallel, set out to structurally characterize it by NMR. Its ß-lactamase activity in relation to the solution structure by NMR is likely to provoke deeper understanding of its mechanism and facilitate the rationalization of other functions of the protein, if any. In this endeavor, we report almost complete sequence-specific backbone (1)H, (13)C and (15)N NMR assignments of UVI31+.

Identification of C-terminal neighbours of amino acid residues without an aliphatic 13Cgamma as an aid to NMR assignments in proteins.

We propose a methodology that uses GFT (3,2)D CB(CACO)NNH experiment to rapidly collect the data and readily identify six amino acid residue types (Ala, Asn, Asp, Cys, Gly and Ser) in any given protein. Further, the experiment can distinguish the redox state of Cys residues. The proposed experiment in its two forms will have wide range of applications in resonance assignment strategies and structure determination of proteins.

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM).

We report almost complete sequence specific 1H, 13C and 15N NMR assignments of a 151-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaM).

Rapid measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides.

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.