Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT.

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

Dynamic Allostery Modulates Catalytic Activity by Modifying the Hydrogen Bonding Network in the Catalytic Site of Human Pin1.

Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a WW domain for substrate recognition and a peptidyl-prolyl isomerase (PPIase) domain. Previous studies of Pin1 showed that physical contact between the domains causes dynamic allostery by reducing conformation dynamics in the catalytic domain, which compensates for the entropy costs of substrate binding to the catalytic site and thus increases catalytic activity. In this study, the S138A mutant PPIase domain, a mutation that mimics the structural impact of the interdomain contact, was demonstrated to display dynamic allostery by rigidification of the α2-α3 loop that harbors the key catalytic residue C113. The reduced dynamics of the α2-α3 loop stabilizes the C113-H59 hydrogen bond in the hydrogen-bonding network of the catalytic site. The stabilized hydrogen bond between C113 and H59 retards initiation of isomerization, which explains the reduced isomerization rate by ~20% caused by the S138A mutation. These results provide new insight into the interdomain allosteric communication of Pin1.

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy.

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.

Allosteric Breakage of the Hydrogen Bond within the Dual-Histidine Motif in the Active Site of Human Pin1 PPIase.

Intimate cooperativity among active site residues in enzymes is a key factor for regulating elaborate reactions that would otherwise not occur readily. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is the phosphorylation-dependent cis-trans peptidyl-prolyl isomerase (PPIase) that specifically targets phosphorylated Ser/Thr-Pro motifs. Residues C113, H59, H157, and T152 form a hydrogen bond network in the active site, as in the noted connection. Theoretical studies have shown that protonation to thiolate C113 leads to rearrangement of this hydrogen bond network, with switching of the tautomeric states of adjacent histidines (H59 and H157) [Barman, A., and Hamelberg, D. (2014) Biochemistry 53, 3839-3850]. This is called the "dual-histidine motif". Here, C113A and C113S Pin1 mutants were found to alter the protonation states of H59 according to the respective residue type replaced at C113, and the mutations resulted in disruption of the hydrogen bond within the dual-histidine motif. In the C113A mutant, H59 was observed to be in exchange between ε- and δ-tautomers, which widened the entrance of the active site cavity, as seen by an increase in the distance between residues A113 and S154. The C113S mutant caused H59 to exchange between the ε-tautomer and imidazolium while not changing the active site structure. Moreover, the imidazole ring orientations of H59 and H157 were changed in the C113S mutant. These results demonstrated that a mutation at C113 modulates the hydrogen bond network dynamics. Thus, C113 acts as a pivot to drive the concerted function among the residues in the hydrogen bond network, as theoretically predicted.

The C113D mutation in human Pin1 causes allosteric structural changes in the phosphate binding pocket of the PPIase domain through the tug of war in the dual-histidine motif.

Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.

Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA.

HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA.