KCTD5 is endowed with large, functionally relevant, interdomain motions

<p>The KCTD family is an emerging class of proteins that are involved in important biological processes whose biochemical and structural properties are rather poorly characterized or even completely undefined. We here used KCTD5, the only member of the family with a known three-dimensional structure, to gain insights into the intrinsic structural stability of the C-terminal domain (CTD) and into the mutual dynamic interplay between the two domains of the protein. Molecular dynamics (MD) simulations indicate that in the simulation timescale (120 ns), the pentameric assembly of the CTD is endowed with a significant intrinsic stability. Moreover, MD analyses also led to the identification of exposed β-strand residues. Being these regions intrinsically sticky, they could be involved in the substrate recognition. More importantly, simulations conducted on the full-length protein provide interesting information of the relative motions between the BTB domain and the CTD of the protein. Indeed, the dissection of the overall motion of the protein is indicative of a large interdomain twisting associated with limited bending movements. Notably, MD data indicate that the entire interdomain motion is pivoted by a single residue (Ser150) of the hinge region that connects the domains. The functional relevance of these motions was evaluated in the context of the functional macromolecular machinery in which KCTD5 is involved. This analysis indicates that the interdomain twisting motion here characterized may be important for the correct positioning of the substrate to be ubiquitinated with respect to the other factors of the ubiquitination machinery.</p

Structural conversion of the transformer protein RfaH: new insights derived from protein structure prediction and molecular dynamics simulations

<div><p>Recent structural investigations have shown that the C-terminal domain (CTD) of the transcription factor RfaH undergoes unique structural modifications that have a profound impact into its functional properties. These modifications cause a complete change in RfaH^CTD topology that converts from an <i>α</i>-hairpin to a <i>β</i>-barrel fold. To gain insights into the determinants of this major structural conversion, we here performed computational studies (protein structure prediction and molecular dynamics simulations) on RfaH^CTD. Although these analyses, in line with literature data, suggest that the isolated RfaH^CTD has a strong preference for the <i>β</i>-barrel fold, they also highlight that a specific region of the protein is endowed with a chameleon conformational behavior. In particular, the Leu-rich region (residues 141–145) has a good propensity to adopt both <i>α</i>-helical and <i>β</i>-structured states. Intriguingly, in the RfaH homolog NusG, whose CTD uniquely adopts the <i>β</i>-barrel fold, the corresponding region is rich in residues as Val or Ile that present a strong preference for the <i>β</i>-structure. On this basis, we suggest that the presence of this Leu-rich element in RfaH^CTD may be responsible for the peculiar structural behavior of the domain. The analysis of the sequences of RfaH family (PfamA code PF02357) unraveled that other members potentially share the structural properties of RfaH^CTD. These observations suggest that the unusual conformational behavior of RfaH^CTD may be rare but not unique.</p></div

The dynamic properties of the Hepatitis C Virus E2 envelope protein unraveled by molecular dynamics

<p>Hepatitis C Virus (HCV) is one of the most persistent human viruses. Although effective therapeutic approaches have been recently discovered, their use is limited by the elevated costs. Therefore, the development of alternative/complementary strategies is an urgent need. The E2 glycoprotein, the most immunogenic HCV protein, and its variants represent natural candidates to achieve this goal. Here we report an extensive molecular dynamics (MD) analysis of the intrinsic properties of E2. Our data provide interesting clues on the global and local intrinsic dynamic features of the protein. Present MD data clearly indicate that E2 combines a flexible structure with a network of covalent bonds. Moreover, the analysis of the two most important antigenic regions of the protein provides some interesting insights into their intrinsic structural and dynamic properties. Our data indicate that a fluctuating β-hairpin represents a populated state by the region E2^412−423. Interestingly, the analysis of the epitope E2^427−446 conformation, that undergoes a remarkable rearrangement in the simulation, has significant similarities with the structure that the E2^430−442 fragment adopts in complex with a neutralizing antibody. Present data also suggest that the strict conservation of Gly436 in E2 protein of different HCV genotypes is likely dictated by structural restraints. Moreover, the analysis of the E2^412−423 flexibility provides insights into the mechanisms that some antibodies adopt to anchor Trp437 that is fully buried in E2. Finally, the present investigation suggests that MD simulations should systematically complement crystallographic studies on flexible proteins that are studied in combination with antibodies.</p

Ibuprofen and Propofol Cobinding Effect on Human Serum Albumin Unfolding in Urea

The unfolding pathway of the defatted human serum albumin (HSA) binding ibuprofen and propofol has been studied by using small-angle X-ray scattering (SAXS) and the support of circular dichroism data. A set of HSA solutions with urea concentrations between 0.00 and 9.00 M was analyzed, and the singular value decomposition method applied to the complete SAXS data set allowed us to distinguish four different states in solution. Besides the native and unfolded forms, two intermediates I1 and I2 have been identified, and the low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. The I1 structure was characterized by only one open domain (domain I, which does not host a binding site for either of the ligands), whereas I2 presents only one closed domain (domain III). A direct comparison with the unfolding pathway of the HSA:Ibu complex (Galantini et al. <i>Biophys. Chem.</i> <b>2010</b>, <i>147</i>, 111–122) pointed out that the presence of propofol as a second ligand, located in subdomain IIIB, leads to the appearance of an intermediate with two closed domains (domains II and III), which are those that accommodate the ligands. Moreover, the equilibrium between I2 and the unfolded form is slightly shifted toward higher urea concentrations. These results suggest that the cobinding significantly hinders the unfolding process

Cullin 3 Recognition Is Not a Universal Property among KCTD Proteins

<div><p>Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6^BTB and KCTD11^BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12^BTB and KCTD15^BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11^BTB and KCTD12^BTB is sufficient to abolish the ability of KCTD11^BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.</p></div

Root mean square fluctuations per residue of KCTD11^BTB and CHIM11/12^BTB.

<p>RMSF values calculated on C^α atoms in the equilibrated region the trajectories (20–100 ns) for the simulations carried out on KCTD11^<b>BTB</b> (A) and CHIM11/12^<b>BTB</b> (B). Secondary structure elements are represented as bars. Helices and strands are colored in blue and red, respectively. In the insets the RMSF values of the α2β3 loops, within the different amino acid sequences, are reported.</p

Biophysical characterization of CHIM11/12^BTB conducted by Far-UV CD spectroscopy (A) and by light scattering (B).

<p>The dashed line in (A) represents the far-UV CD spectrum of KCTD11^<b>BTB</b>. The experiments were carried out in a 20mM sodium phosphate buffer (pH 7.5) containing 2 mM DTT.</p

Cul3 binding site in the average MD structures of KCTD11^BTB and CHIM11/12^BTB.

<p>KCTD11^<b>BTB</b>, CHIM11/12^<b>BTB</b>, Cul3^<b>NTD</b> are represented in blue, red and green, respectively. For clarity, a single chain of the tetramers is highlighted.</p

Quantification of Cul3-KCTDs binding by Isothermal Titration Calorimetry.

<p>ITC experiments were performed by titrating KCTD6^<b>BTB</b> (A), KCTD11^<b>BTB</b> (B), KCTD12^<b>BTB</b> (C) with Cul3^<b>NTD</b>. For KCTD15^<b>BTB</b> the ITC experiment was reversed by titrating Cul3^<b>NTD</b> with KCTD15^<b>BTB</b> (D). The top and bottom panels report raw and integrated data, respectively.</p

Percentages of sequence identity and number of aligned residues of the BTB domains of selected KCTD members are reported on the right and left side of the diagonal, respectively.

<p>In addition to the proteins here characterized (KCTD6, KCTD11, KCTD12, and KCTD15), we included into the comparison representative members (KCTD5, KCTD7, KCTD13, BTBD10 and SHKBP1) of KCTD subgroups whose interaction with Cul3 has been experimentally demonstrated.</p><p>Percentages of sequence identity and number of aligned residues of the BTB domains of selected KCTD members are reported on the right and left side of the diagonal, respectively.</p