NMR Insights into Folding and Self-Association of Plasmodium falciparum P2

The eukaryotic 60S-ribosomal stalk is composed of acidic ribosomal proteins (P1 and P2) and neutral protein P0, which are thought to be associated as a pentameric structure, [2P1, 2P2, P0]. Plasmodium falciparum P2 (PfP2) appears to play additional non-ribosomal functions associated with its tendency for homo-oligomerization. Recombinant bacterially expressed PfP2 protein also undergoes self-association, as shown by SDS-PAGE analysis and light scattering studies. Secondary structure prediction algorithms predict the native PfP2 protein to be largely helical and this is corroborated by circular dichroism investigation. The 1H-15N HSQC spectrum of native P2 showed only 43 cross peaks compared to the expected 138. The observed peaks were found to belong to the C-terminal region, suggesting that this segment is flexible and solvent exposed. In 9 M urea denaturing conditions the chain exhibited mostly non-native β structural propensity. 15N Relaxation data for the denatured state indicated substantial variation in ms-µs time scale motion along the chain. Average area buried upon folding (AABUF) calculations on the monomer enabled identification of hydrophobic patches along the sequence. Interestingly, the segments of slower motion in the denatured state coincided with these hydrophobic patches, suggesting that in the denatured state the monomeric chain undergoes transient hydrophobic collapse. The implications of these results for the folding mechanism and self-association of PfP2 are discussed

Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated

Molecular modelling reveals how abundance of α4 sub-type in synaptic GABARA receptor can lead to refractoriness toward GABA and BZ-type drugs

Epilepsy is a complex neurological disorder with genetic and acquired causes, and the drugs presently used to treat epilepsy are not effective in about 30% of the cases. Identification of the molecular mechanisms of resistance will help in the development of newer molecules for treatment. Recent clinical data indicate increased expression of α4- and γ2-containing synaptic GABARA receptors in patients of focal cortical dysplasia (FCD), which is associated with refractory epilepsy pathology. We have investigated, by molecular modelling and docking, the structure and ligand-binding efficiency of the α4-containing hetero-pentameric synaptic GABARA receptor. Though the overall conformation is similar to that of the α1-containing receptor, local conformational changes are seen due to differences between aligned α1 and α4 sub-type residues. The overlaps ALA209(α1)/PRO215(α4) and PHE73(α1)/TYR79(α4) have together caused conformational changes in ARG100(α4) (aligned with ARG94 in α1) thereby affecting key hydrogen bonding interactions with the inhibitory neurotransmitter GABA. This may influence the nature of seizures as strength of GABA-binding is known to affect the nature of Inhibitory Post-Synaptic Currents (IPSCs) from GABAergic neurons. The residue ARG135 (α4) aligns with the residue HIS129 (α1) in the benzodiazapine binding pocket. Molecular modelling also shows that a steric clash between benzodiazapine-type (BZ-type) drugs and ARG135 would reduce the binding of BZ-type drugs to α4-containing receptor. These two findings rationalize the observed association between over-expression of α4-containing synaptic GABARA receptors and refractory epilepsy pathology in FCD. The accurate three-dimensional geometry of the receptor-drug complex made available by these modelling studies will help in designing effective drugs

Identification of 3'-UTR single nucleotide variants and prediction of select protein imbalance in mesial temporal lobe epilepsy patients.

The genetic influence in epilepsy, characterized by unprovoked and recurrent seizures, is through variants in genes critical to brain development and function. We have carried out variant calling in Mesial Temporal Lobe Epilepsy (MTLE) patients by mapping the RNA-Seq data available at SRA, NCBI, USA onto human genome assembly hg-19. We have identified 1,75,641 SNVs in patient samples. These SNVs are distributed over 14700 genes of which 655 are already known to be associated with epilepsy. Large number of variants occur in the 3'-UTR, which is one of the regions involved in the regulation of protein translation through binding of miRNAs and RNA-binding proteins (RBP). We have focused on studying the structure-function relationship of the 3'-UTR SNVs that are common to at-least 10 of the 35 patient samples. For the first time we find SNVs exclusively in the 3'-UTR of FGF12, FAR1, NAPB, SLC1A3, SLC12A6, GRIN2A, CACNB4 and FBXO28 genes. Structural modelling reveals that the variant 3'-UTR segments possess altered secondary and tertiary structures which could affect mRNA stability and binding of RBPs to form proper ribonucleoprotein (RNP) complexes. Secondly, these SNVs have either created or destroyed miRNA-binding sites, and molecular modeling reveals that, where binding sites are created, the additional miRNAs bind strongly to 3'-UTR of only variant mRNAs. These two factors affect protein production thereby creating an imbalance in the amounts of select proteins in the cell. We suggest that in the absence of missense and nonsense variants, protein-activity imbalances associated with MTLE patients can be caused through 3'-UTR variants in relevant genes by the mechanisms mentioned above. 3'-UTR SNV has already been identified as causative variant in the neurological disorder, Tourette syndrome. Inhibition of these miRNA-mRNA bindings could be a novel way of treating drug-resistant MTLE patients. We also suggest that joint occurrence of these SNVs could serve as markers for MTLE. We find, in the present study, SNV-mediated destruction of miRNA binding site in the 3'-UTR of the gene encoding glutamate receptor subunit, and, interestingly, overexpression of one of this receptor subunit is also associated with Febrile Seizures

Sequence effects in structures of the dinucleotides d-pApT AND d-pTpA

The H1',H2' and H2″ regions of the 270-MHz PMR spectra of two deoxydinucleotides, d-pTpA and d-pApT, have been analyzed. The coupling constants in the sugar ring indicate that both A and T sugars have a tendency to acquire 2E conformations. There is also a marginal difference in the 2E populations of the T sugar in the two dinucleotides. The trends in the chemical shifts of base protons indicate different stacking of the bases in d-pApT and d-pTpA. The sequence effects on base stacking and pentose conformation are discussed

Sequence effects in structures of the dinucleotides d-pApT AND d-pTpA

The H1', H2' and H" regions of the 270-MHz PMR spectra of two deoxydinucleotides, d-pTpA and d-pApT, have been analyzed. The coupling constants in the sugar ring indicate that both A and T sugars have a tendency to acquire 2E conformations. There is also a marginal difference in the 2E populations of the T sugar in the two dinucleotides. The trends in the chemical shifts of base protons indicate different stacking of the bases in d-pApT and d-pTpA. The sequence effects on base stacking and pentose conformation are discussed

Secondary structure predictions.

<p>Summary of structure prediction details of PfP2 using six different programs. Cylinders show α-helical regions, arrows show β sheet and lines show random coils.</p

NMR secondary chemical shifts.

<p>(A) ΔH^α secondary chemical shift (B) ΔC^α secondary chemical shift for (C) ΔCO secondary chemical shift of 9 M urea denatured state of PfP2 at pH 5.6 and 27°C. The secondary structural propensities are indicated in each box; arrow indicates β-propensity. Shaded grey region represents a stretch that is coming from the vector and is not part of PfP2.</p

Insights into folding and self-association.

<p>(A) Plots of calculated J(0) values at 800 MHz and AABUF (average area buried upon folding) for 9 M urea denatured state of PfP2. The AABUF were calculated with the expasy tool protscale [<a href="https://www.us.expasy.org/tools/protscale.html" target="_blank">https://www.us.expasy.org/tools/protscale.html</a>]. The predicted secondary structure is shown on top (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036279#pone-0036279-g003" target="_blank">Figure 3</a>). The regions with increased AABUF values have been marked with black bars and those with reduced flexibility have been marked with grey bars. Shaded grey region represents a stretch that is coming from the vector and is not part of PfP2 (B) Schematic Representation of Hydrophobic collapse model of folding and subsequent association of PfP2. (C) The electrostatic potential (isocontour value ±10 KT/e) surface of PfP2 calculated using a theoretically generated 3D structure of the protein with surface amino acid charges are depicted in red (negative charge) and blue (positive charge). Neutral elements are depicted in white color. This clearly indicates that the surface is highly hydrophobic in nature.</p

NMR assignments in urea denatured PfP2.

<p>(A) 2D ¹H,¹⁵N HSQC spectrum of PfP2 in 9 M urea at pH 5.6 and 27°C. Residue specific assignment for each peak is marked on the spectrum (B) Summary of sequential assignment. In A, Peaks from the vector are marked in red. Out of 112 protein residues (138 minus 26 which derive from the vector) excluding the two proline residues, 104 have been sequence-specifically assigned; the Vector peaks have also been assigned. Six peaks could not be assigned because of not availability of sequential connectivities. The spectra contained some additional peaks of less intensity which also showed some sequential connectivity. These are more likely due to existence of some other conformation in slow exchange. Assignment is submitted in BMRB under the accession code 17616.</p