Structural dynamics in the La-module of La-related proteins

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Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases

RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2~Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2~Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5~Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7~Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism

Shedding Light on the Interaction between TMPyP4 and Human Telomeric Quadruplexes

The nature of the binding mode and stoichiometry of the TMPyP4 cationic porphyrin to G-quadruplex structures continues to be controversial, with no consensus model to date, especially for intramolecular G-quadruplexes from human telomeric sequences. Those sequences possess intricate polymorphism in solution that appears to be reduced under molecular crowding conditions in which the parallel structure appears to be the most populated one. We have performed a systematic study, in dilute solution and under molecular crowding conditions, of the binding reactions between TMPyP4 and four G-quadruplexes formed by different truncations of human telomeric DNA, with 5′- or 3′-flanking bases, using isothermal titration calorimetry and circular dichroism. The results clearly indicate that all of these G-quadruplexes are able to bind up to four TMPyP4 molecules. CD studies show that interaction with TMPyP4 promotes the conversion of the hybrid structures to an antiparallel conformation in dilute solution, while under molecular crowding conditions the interaction does not promote any conformational change. ITC reveals in both cases that the binding process comprises two sequential events, a first in which one molecule of TMPyP4 interacts with the quadruplex structures and a second in which three other molecules bind to the structures. The selectivity of TMPyP4 for the quadruplex relative to duplex DNA was also investigated under molecular crowding conditions showing that TMPyP4 has enhanced selectivity for quadruplex DNA compared to the duplex structure. This finding reinforces the potential applications of TMPyP4

Shedding Light on the Interaction between TMPyP4 and Human Telomeric Quadruplexes

The nature of the binding mode and stoichiometry of the TMPyP4 cationic porphyrin to G-quadruplex structures continues to be controversial, with no consensus model to date, especially for intramolecular G-quadruplexes from human telomeric sequences. Those sequences possess intricate polymorphism in solution that appears to be reduced under molecular crowding conditions in which the parallel structure appears to be the most populated one. We have performed a systematic study, in dilute solution and under molecular crowding conditions, of the binding reactions between TMPyP4 and four G-quadruplexes formed by different truncations of human telomeric DNA, with 5′- or 3′-flanking bases, using isothermal titration calorimetry and circular dichroism. The results clearly indicate that all of these G-quadruplexes are able to bind up to four TMPyP4 molecules. CD studies show that interaction with TMPyP4 promotes the conversion of the hybrid structures to an antiparallel conformation in dilute solution, while under molecular crowding conditions the interaction does not promote any conformational change. ITC reveals in both cases that the binding process comprises two sequential events, a first in which one molecule of TMPyP4 interacts with the quadruplex structures and a second in which three other molecules bind to the structures. The selectivity of TMPyP4 for the quadruplex relative to duplex DNA was also investigated under molecular crowding conditions showing that TMPyP4 has enhanced selectivity for quadruplex DNA compared to the duplex structure. This finding reinforces the potential applications of TMPyP4

Structural dynamics in the La-module of La-related proteins

The La-related proteins (LaRPs) are a superfamily of eukaryotic RNA-binding proteins with important and varied roles. To understand LaRP functions it is essential to unravel the divergent features responsible for their RNA target selectivity, which underlie their distinct identities and cellular roles. LaRPs are built on a common structural module called the ‘La-module’ that acts as a main locus for RNA recognition. The La-module is comprised of two tethered domains whose relative structural and dynamic interplay has been proposed to regulate RNA-target selection, albeit the mechanistic underpinning of this recognition remains to be elucidated. A main unsolved conundrum is how conserved La-modules across LaRPs are able to bind to extremely diverse RNA ligands. In this work, we employed Small Angle X-ray Scattering (SAXS) to investigate several human LaRP La-modules in the absence and, where applicable, in the presence of their RNA target, with the aim to explore the structural dynamics of their RNA recognition and provide information on the architectural landscape accessible to these proteins. Integration of these SAXS experiments with prior X-ray crystallography and NMR data suggests that RNA binding is generally accompanied by a compaction and loss of flexibility of the La-module. Nonetheless, the La-modules appear to experience a considerably different degree of inherent flexibility in their apo state. Furthermore, although they all exist in discrete subsets of accessible populations in equilibrium, these vary from LaRP to LaRP and can be either extended or compact. We propose that these divergent features may be critical for RNA substrate discrimination.</p

Domain organisation and expression screen of human NLRP1.

(A) Human NLRP1 domain organisation; black lines indicate some of the key residues reported to be important for protein function. K340 and E414 belong to the Walker A and Walker B motifs, respectively and are important for ATP processing. H623 is a conserved residue across all the NLRs, the correspondent residues in NLRC4 and in Apaf1 are involved in stabilising the ADP-bound conformation. H1190, F1216 and S1217 are reported to be important for the auto-proteolysis of the FIIND domain. (B) Schematic representation of the soluble constructs produced in insect cells. The boundaries of each construct are indicated on the left. (C) SDS-gels of the recombinant proteins from insect cells after the first metal affinity purification step. A black star indicates the protein of interest and a black arrow heads indicates proteolytic degradation products.</p

SAXS-derived envelope of NLRP1(227–990) and comparison to the open and closed conformations of NLRC4.

(A) SAXS-derived envelope for NLRP1(227–990) calculated from the SAXS data obtained at 2 mg/mL, the maximum dimension (Dmax) and the width of the upper and lower lobes are also reported. (B and C) Structural fitting of the structures of NLRC4 spanning residues 93 to 793 in an open conformation (PDB 3jbl, χ2 of 1.37, in magenta) and in a closed conformation (PDB 4kxf, χ2 of 1.97, in yellow) into the NLRP1 envelope.</p

The biophysical characterisation and SAXS analysis of human NLRP1 uncover a new level of complexity of NLR proteins

NOD-like receptors represent an important class of germline-encoded pattern recognition receptors that play key roles in the regulation of inflammatory signalling pathways. They function as danger sensors and initiate inflammatory responses and the production of cytokines. Since NLR malfunction results in chronic inflammation and auto-immune diseases, there is a great interest in understanding how they work on a molecular level. To date, a lot of insight into the biological functions of NLRs is available but biophysical and structural studies have been hampered by the difficulty to produce soluble and stable recombinant NLR proteins. NLRP1 is an inflammasome forming NLR that is believed to be activated by binding to MDP and induces activation of caspase 1. Here, we report the identification of a soluble fragment of NLRP1 that contains the NACHT oligomerization domain and the putative MDP-sensing LRR domain. We describe the biophysical and biochemical characterization of this construct and a SEC-SAXS analysis that allowed the calculation of a low resolution molecular envelope. Our data indicate that the protein is constitutively bound to ATP with a negligible ability to hydrolyse the triphosphate nucleotide and that it adopts a monomeric extended conformation that is reminiscent of the structure adopted by NLRC4 in the inflammasome complex. Furthermore, we show that the presence of MDP is not sufficient to promote self-oligomerization of the NACHT-LRR fragment suggesting that MDP may either bind to regions outside the NACHT-LRR module or that it may not be the natural ligand of NLRP1. Taken together, our data suggest that the NLRP1 mechanism of action differs from that recently reported for other NLRs

Preliminary biophysical characterisation of recombinant NLRP1(227–990).

<p>(A) Size exclusion chromatography profile of the construct NLRP1(227–990) on a S200 XK16/60 column. Black arrow heads indicate the retention volumes of molecular weight standards. The sample migrates as a single species with an apparent molecular weight between 44–158 kDa. (B) Far-UV circular dichroism spectrum of NLRP1(227–990). (C) Thermal unfolding of NLRP1(227–990) obtained by following the CD signal at 222 nm as a function of the temperature increased at 2°C/min, the value of the mid-point transition is 54.3±0.5°C.</p

ATP hydrolysis and oligomerization assay of NLRP1(227–990).

<p>(A) HPLC chromatogram showing the profile of a solution of ADP and ATP standards run on an ion exchange Partisil SAX column (grey solid line), retention times for ADP 2.81 minutes and ATP 6.12 minutes. The sample prepared by unfolding of NLRP1(227–990) contained mainly ATP (>85%) (black solid line). The peak with a retention time of about 2 minutes is background from the buffer. (B) Malachite green assay performed on a solution of NLRP1(227–990) in the presence of ATP 1mM (black squares) and in the presence of ATP 1mM and MDP 0.1mg/mL (black triangles). In both cases the concentration of protein was 0.1 mg/mL and the experiment was performed at room temperature for 1 hour. A blank experiment, without NLRP1 and MDP (black diamonds) and a positive control with DnaK (black stars) were also performed. (C) Size exclusion chromatography (Superose 6 10/300 column) of NLRP1(227–990) in absence (black solid line), in presence of MDP and ATP (grey dotted line) and in presence of MDP and a non-hydrolysable ATP analogue (grey solid line). Black arrow heads indicate the retention volumes of molecular weight standards.</p