We designed a minimalistic zinc(II)-binding peptide featuring the Cys2His2 zinc-finger motif. To this aim, several tens of thousands of (His/Cys)-Xn-(His/Cys) protein fragments (n=2-20) were first extracted from the 3D protein structures deposited in Protein Data Bank (PDB). Based on geometrical constraints positioning two Cys (C) and two His (H) side chains at the vertices of a tetrahedron, approximately 22 000 sequences of the (H/C)-Xi-(H/C)-Xj-(H/C)-Xk-(H/C) type, satisfying Nmetal-binding H=Nmetal-binding C=2, were processed. Several other criteria, such as the secondary structure content and predicted fold stability, were then used to select the best candidates. To prove the viability of the computational design experimentally, three peptides were synthesized and subjected to isothermal calorimetry (ITC) measurements to determine the binding constants with Zn2+, including the entropy and enthalpy terms. For the strongest Zn2+ ions binding peptide, P1, the dissociation constant was shown to be in the nanomolar range (KD=~220 nM; corresponding to ΔGbind=-9.1 kcal mol-1). In addition, ITC showed that the [P1 : Zn2+] complex forms in 1 : 1 stoichiometry and two protons are released upon binding, which suggests that the zinc coordination involves both cysteines. NMR experiments also indicated that the structure of the [P1 : Zn2+] complex might be quite similar to the computationally predicted one. In summary, our proof-of-principle study highlights the usefulness of our computational protocol for designing novel metal-binding peptides.

• Klíčová slova
• Computer design, Isothermal calorimetry, Metal-binding peptide, NMR, QM modeling, Zinc(II),
• MeSH
• molekulární modely MeSH
• peptidy * chemie   metabolismus   chemická syntéza   MeSH
• sekvence aminokyselin MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• zinek * chemie   metabolismus   MeSH
• zinkové prsty MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy * MeSH
• zinek * MeSH

RNA recognition motifs (RRMs) are a key class of proteins that primarily bind single-stranded RNAs. In this study, we applied standard atomistic molecular dynamics simulations to obtain insights into the intricate binding dynamics between uridine-rich RNAs and TbRGG2 RRM using the recently developed OL3-Stafix AMBER force field, which improves the description of single-stranded RNA molecules. Complementing structural experiments that unveil a primary binding mode with a single uridine bound, our simulations uncover two supplementary binding modes in which adjacent nucleotides encroach upon the binding pocket. This leads to a unique molecular mechanism through which the TbRGG2 RRM is capable of rapidly transitioning the U-rich sequence. In contrast, the presence of non-native cytidines induces stalling and destabilization of the complex. By leveraging extensive equilibrium dynamics and a large variety of binding states, TbRGG2 RRM effectively expedites diffusion along the RNA substrate while ensuring robust selectivity for U-rich sequences despite featuring a solitary binding pocket. We further substantiate our description of the complex dynamics by simulating the fully spontaneous association process of U-rich sequences to the TbRGG2 RRM. Our study highlights the critical role of dynamics and auxiliary binding states in interface dynamics employed by RNA-binding proteins, which is not readily apparent in traditional structural studies but could represent a general type of binding strategy employed by many RNA-binding proteins.

• MeSH
• konformace nukleové kyseliny MeSH
• motiv rozpoznávající RNA * MeSH
• proteiny vázající RNA * chemie   metabolismus   MeSH
• RNA * metabolismus   chemie   MeSH
• simulace molekulární dynamiky * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny vázající RNA * MeSH
• RNA * MeSH

The N-terminal RNA recognition motif domain (RRM1) of polypyrimidine tract binding protein (PTB) forms an additional C-terminal helix α3, which docks to one edge of the β-sheet upon binding to a stem-loop RNA containing a UCUUU pentaloop. Importantly, α3 does not contact the RNA. The α3 helix therefore represents an allosteric means to regulate the conformation of adjacent domains in PTB upon binding structured RNAs. Here we investigate the process of dynamic adaptation by stem-loop RNA and RRM1 using NMR and MD in order to obtain mechanistic insights on how this allostery is achieved. Relaxation data and NMR structure determination of the free protein show that α3 is partially ordered and interacts with the domain transiently. Stem-loop RNA binding quenches fast time scale dynamics and α3 becomes ordered, however microsecond dynamics at the protein-RNA interface is observed. MD shows how RRM1 binding to the stem-loop RNA is coupled to the stabilization of the C-terminal helix and helps to transduce differences in RNA loop sequence into changes in α3 length and order. IRES assays of full length PTB and a mutant with altered dynamics in the α3 region show that this dynamic allostery influences PTB function in cultured HEK293T cells.

• MeSH
• alosterická regulace MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• motiv rozpoznávající RNA MeSH
• protein vázající polypyrimidinové úseky RNA * metabolismus   chemie   MeSH
• proteinové domény MeSH
• RNA * chemie   metabolismus   MeSH
• sbalování proteinů MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů * MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protein vázající polypyrimidinové úseky RNA * MeSH
• RNA * MeSH

Canonical Wnt signaling is essential for a plethora of biological processes ranging from early embryogenesis to aging. Malfunctions of this crucial signaling pathway are associated with various developmental defects and diseases, including cancer. Although TCF/LEF transcription factors (TCF/LEFs) are known to be essential for this pathway, the regulation of their intracellular levels is not completely understood. Here, we show that the lysine demethylase KDM2A promotes the proteasomal destabilization of TCF/LEFs independently of its demethylase domain. We found that the KDM2A-mediated destabilization of TCF/LEFs is dependent on the KDM2A zinc finger CXXC domain. Furthermore, we identified the C-terminal region of TCF7L2 and the CXXC domain of KDM2A as the domains responsible for the interaction between the two proteins. Our study is also the first to show that endogenous TCF/LEF proteins undergo KDM2A-mediated proteasomal degradation in a neddylation-dependent manner. Here, we reveal a completely new mechanism that affects canonical Wnt signaling by regulating the levels of TCF/LEF transcription factors through their KDM2A-promoted proteasomal degradation.

• Klíčová slova
• KDM2A, TCF/LEF, canonical Wnt signaling, neddylation, ubiquitination,
• MeSH
• beta-katenin * metabolismus   MeSH
• lysin * MeSH
• signální dráha Wnt MeSH
• zinkové prsty MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin * MeSH
• lysin * MeSH

Recognition of single-stranded RNA (ssRNA) by RNA recognition motif (RRM) domains is an important class of protein-RNA interactions. Many such complexes were characterized using nuclear magnetic resonance (NMR) and/or X-ray crystallography techniques, revealing ensemble-averaged pictures of the bound states. However, it is becoming widely accepted that better understanding of protein-RNA interactions would be obtained from ensemble descriptions. Indeed, earlier molecular dynamics simulations of bound states indicated visible dynamics at the RNA-RRM interfaces. Here, we report the first atomistic simulation study of spontaneous binding of short RNA sequences to RRM domains of HuR and SRSF1 proteins. Using a millisecond-scale aggregate ensemble of unbiased simulations, we were able to observe a few dozen binding events. HuR RRM3 utilizes a pre-binding state to navigate the RNA sequence to its partially disordered bound state and then to dynamically scan its different binding registers. SRSF1 RRM2 binding is more straightforward but still multiple-pathway. The present study necessitated development of a goal-specific force field modification, scaling down the intramolecular van der Waals interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens up a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes, and future perspectives of such research are discussed.

• MeSH
• HuR protein metabolismus   MeSH
• motiv rozpoznávající RNA genetika   MeSH
• proteiny vázající RNA * metabolismus   MeSH
• RNA * chemie   MeSH
• RRM proteiny metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HuR protein MeSH
• proteiny vázající RNA * MeSH
• RNA * MeSH
• RRM proteiny MeSH

RNA-protein complexes use diverse binding strategies, ranging from structurally well-defined interfaces to completely disordered regions. Experimental characterization of flexible segments is challenging and can be aided by atomistic molecular dynamics (MD) simulations. Here, we used an extended set of microsecond-scale MD trajectories (400 μs in total) to study two FUS-RNA constructs previously characterized by nuclear magnetic resonance (NMR) spectroscopy. The FUS protein contains a well-structured RNA recognition motif domain followed by a presumably disordered RGG tail that binds RNA stem-loop hairpins. Our simulations not only provide several suggestions complementing the experiments but also reveal major methodological difficulties in studies of such complex RNA-protein interfaces. Despite efforts to stabilize the binding via system-specific force-field adjustments, we have observed progressive distortions of the RNA-protein interface inconsistent with experimental data. We propose that the dynamics is so rich that its converged description is not achievable even upon stabilizing the system. Still, after careful analysis of the trajectories, we have made several suggestions regarding the binding. We identify substates in the RNA loops, which can explain the NMR data. The RGG tail localized in the minor groove remains disordered, sampling countless transient interactions with the RNA. There are long-range couplings among the different elements contributing to the recognition, which can lead to allosteric communication throughout the system. Overall, the RNA-FUS systems form dynamical ensembles that cannot be fully represented by single static structures. Thus, albeit imperfect, MD simulations represent a viable tool to investigate dynamic RNA-protein complexes.

• MeSH
• molekulární konformace MeSH
• motiv rozpoznávající RNA * MeSH
• proteiny chemie   MeSH
• RNA chemie   MeSH
• simulace molekulární dynamiky * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH
• RNA MeSH

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, ∆1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1-854 hTRPA1, also lacking the S1-S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

• MeSH
• alanin MeSH
• ankyrinová repetice * MeSH
• kationtový kanál TRPA1 genetika   metabolismus   MeSH
• lidé MeSH
• tryptofan MeSH
• vnímání teploty MeSH
• vysoká teplota * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• kationtový kanál TRPA1 MeSH
• TRPA1 protein, human MeSH Prohlížeč
• tryptofan MeSH

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

• Klíčová slova
• ATM, ATR, DNA damage response pathway, HBV core protein, serine phosphorylation,
• MeSH
• aminokyselinové motivy MeSH
• ATM protein metabolismus   MeSH
• buňky Hep G2 MeSH
• checkpoint kinasa 2 metabolismus   MeSH
• cytoplazma metabolismus   virologie   MeSH
• etoposid farmakologie   MeSH
• fosforylace MeSH
• hepatitida B - antigeny e metabolismus   MeSH
• hepatocyty virologie   MeSH
• lidé MeSH
• peroxid vodíku farmakologie   MeSH
• poškození DNA * MeSH
• proteiny virového jádra chemie   metabolismus   MeSH
• replikace viru účinky léků   MeSH
• serin metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• virové regulační a přídatné proteiny genetika   metabolismus   MeSH
• virus hepatitidy B účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• checkpoint kinasa 2 MeSH
• CHEK2 protein, human MeSH Prohlížeč
• etoposid MeSH
• hepatitida B - antigeny e MeSH
• hepatitis B virus X protein MeSH Prohlížeč
• peroxid vodíku MeSH
• proteiny virového jádra MeSH
• serin MeSH
• trans-aktivátory MeSH
• virové regulační a přídatné proteiny MeSH

The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.

• Klíčová slova
• 9aaTAD, E2A, KIX, MLL, activation domain, p53,
• MeSH
• aminokyselinové motivy MeSH
• histonlysin-N-methyltransferasa chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   metabolismus   MeSH
• NF-kappa B chemie   metabolismus   MeSH
• protein vázající CREB chemie   metabolismus   MeSH
• protoonkogenní protein MLL chemie   metabolismus   MeSH
• transkripční faktory bHLH chemie   metabolismus   MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histonlysin-N-methyltransferasa MeSH
• KMT2A protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• NF-kappa B MeSH
• protein vázající CREB MeSH
• protoonkogenní protein MLL MeSH
• TCF3 protein, human MeSH Prohlížeč
• TP53 protein, human MeSH Prohlížeč
• transkripční faktory bHLH MeSH
• transkripční faktory MeSH

A high level of nucleolin (NCL) expression is often associated with a poor prognosis of patients with lung cancer (LC), suggesting that NCL can be used as a possible biomarker. NCL has been shown to display a marked preference for the binding to G-quadruplexes (G4). Here, we investigate the formation of an RNA quadruplex structure in a sequence found in the human precursor pre-MIR150 with the potential to recognize NCL. Circular dichroism (CD) spectra of pre-MIR150 G4-forming sequence (designated by rG4) indicate the formation of a parallel quadruplex structure in KCl or when complexed with the well-known G4 ligand PhenDC3. The thermal stability of rG4 is very high, and further increases in the presence of PhenDC3. The binding affinities of rG4 to PhenDC3 and NCL RBD1,2 are similar with KD values in the nanomolar range. PAGE results suggest the formation of a ternary quadruplex-ligand-protein complex (rG4-PhenDC3-NCL RBD1,2), indicative that PhenDC3 does not prevent the binding of rG4 to NCL RBD1,2. Finally, rG4 can recognize NCL-positive cells and, when fluorescently labeled, can be used as a probe for this protein. ELISA experiments indicate altered NCL expression patterns in liquid biopsies of LC patients in a non-invasive manner, potentially helping the diagnosis, prognosis, and patient response to treatment. Hence, labeled rG4 could be used as a detection probe of LC in liquid biopsies.

• Klíčová slova
• Liquid biopsies, Lung Cancer, Molecular recognition, Nucleolin, pre-MIR150 G-quadruplex forming sequence,
• MeSH
• aminokyselinové motivy fyziologie   MeSH
• dospělí MeSH
• fosfoproteiny biosyntéza   genetika   MeSH
• G-kvadruplexy * MeSH
• genový targeting metody   MeSH
• kultivované buňky MeSH
• leukocyty mononukleární metabolismus   MeSH
• lidé MeSH
• nádory plic genetika   metabolismus   terapie   MeSH
• nukleolin MeSH
• proteiny vázající RNA biosyntéza   genetika   MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• proteiny vázající RNA MeSH