The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of mA, the most abundant RNA modification. RBM15 positively regulates mA levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes

Investigation of Atomic Level Patterns in Protein—Small Ligand Interactions

BACKGROUND: Shape complementarity and non-covalent interactions are believed to drive protein-ligand interaction. To date protein-protein, protein-DNA, and protein-RNA interactions were systematically investigated, which is in contrast to interactions with small ligands. We investigate the role of covalent and non-covalent bonds in protein-small ligand interactions using a comprehensive dataset of 2,320 complexes. METHODOLOGY AND PRINCIPAL FINDINGS: We show that protein-ligand interactions are governed by different forces for different ligand types, i.e., protein-organic compound interactions are governed by hydrogen bonds, van der Waals contacts, and covalent bonds; protein-metal ion interactions are dominated by electrostatic force and coordination bonds; protein-anion interactions are established with electrostatic force, hydrogen bonds, and van der Waals contacts; and protein-inorganic cluster interactions are driven by coordination bonds. We extracted several frequently occurring atomic-level patterns concerning these interactions. For instance, 73% of investigated covalent bonds were summarized with just three patterns in which bonds are formed between thiol of Cys and carbon or sulfur atoms of ligands, and nitrogen of Lys and carbon of ligands. Similar patterns were found for the coordination bonds. Hydrogen bonds occur in 67% of protein-organic compound complexes and 66% of them are formed between NH- group of protein residues and oxygen atom of ligands. We quantify relative abundance of specific interaction types and discuss their characteristic features. The extracted protein-organic compound patterns are shown to complement and improve a geometric approach for prediction of binding sites. CONCLUSIONS AND SIGNIFICANCE: We show that for a given type (group) of ligands and type of the interaction force, majority of protein-ligand interactions are repetitive and could be summarized with several simple atomic-level patterns. We summarize and analyze 10 frequently occurring interaction patterns that cover 56% of all considered complexes and we show a practical application for the patterns that concerns interactions with organic compounds

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay

Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol

In a crystal structure of the amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG), the biologically active estrogen metabolite, 2-methoxyestradiol (2-MeOE2), binds in the same orientation as estradiol. The high affinity of SHBG for 2-MeOE2 relies primarily on hydrogen bonding between the hydroxyl at C-3 of 2-MeOE2 and Asp65 and an interaction between the methoxy group at C-2 and the amido group of Asn82. Accommodation of the 2-MeOE2 methoxy group causes an outward displacement of residues Ser128-Pro130, which appears to disorder and displace the loop region (Leu131-His136) that covers the steroid-binding site. This could influence the binding kinetics of 2-MeOE2 and/or facilitate ligand-dependent interactions between SHBG and other proteins. Occupancy of a zinc-binding site reduces the affinity of SHBG for 2-MeOE2 and estradiol in the same way. The higher affinity of SHBG for estradiol derivatives with a halogen atom at C-2 is due to either enhanced hydrogen bonding between the hydroxyl at C-3 and Asp65 (2-fluoroestradiol) or accommodation of the functional group at C-2 (2-bromoestradiol), rather than an interaction with Asn82. By contrast, the low affinity of SHBG for 2-hydroxyestradiol can be attributed to intra-molecular hydrogen bonding between the hydroxyls in the aromatic steroid ring A, which generates a steric clash with the amido group of Asn82. Understanding how C-2 derivatives of estradiol interact with SHBG could facilitate the design of biologically active synthetic estrogens

Molecular mimicry enables competitive recruitment by a natively disordered protein

We report the crystal structure of the Escherichia coli TolB-Pal complex, a protein−protein complex involved in maintaining the integrity of the outer membrane (OM) in all Gram-negative bacteria that is parasitized by colicins (protein antibiotics) to expedite their entry into cells. Nuclease colicins competitively recruit TolB using their natively disordered regions (NDRs) to disrupt its complex with Pal, which is thought to trigger translocation of the toxin across a locally destabilized OM. The structure shows induced-fit binding of peptidoglycan-associated lipoprotein (Pal) to the β-propeller domain of TolB causing the N-terminus of one of its α-helices to unwind and several residues to undergo substantial changes in conformation. The resulting interactions with TolB are known to be essential for the stability of the complex and the bacterial OM. Structural comparisons with a TolB-colicin NDR complex reveal that colicins bind at the Pal site, mimicking rearranged Pal residues while simultaneously appearing to block induced-fit changes in TolB. The study therefore explains how colicins recruit TolB in the bacterial periplasm and highlights a novel binding mechanism for a natively disordered protein

Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer

Human sex hormone-binding globulin (SHBG) transports sex steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its steroid binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single steroid-binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and steroid-binding sites are distinct and that both monomers within a homodimeric complex are capable of binding steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a steroid-binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical steroid binding capacities. These data indicate that both subunits of the SHBG homodimer bind steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold

Resolution of a disordered region at the entrance of the human sex hormone-binding globulin steroid-binding site

The crystal structure of human sex hormone-binding globulin (SHBG) has revealed how 5{alpha}-dihydrotestosterone intercalates between the two seven-stranded {beta}-sheets of its amino-terminal laminin G-like domain. However, a region of disorder (residues 130 to 135 of SHBG) was identified together with a zinc-binding site in immediate proximity to the steroid. It has been important to resolve the structure of this region because previous studies have suggested that these residues may contribute to steroid binding directly. Here, we present the 2.35 A and 1.7 A crystal structures of the amino-terminal LG domain of SHBG obtained from a tetragonal crystal form and by EDTA-soaking of a trigonal crystal form, respectively. In both of these new structures, residues Pro130 to Arg135 are now clearly visible. Substitution of the two residues (Leu131Gly and Lys134Ala) pointing towards the steroid has shown that only Leu131 contributes significantly to steroid binding. Rather than covering the steroid-binding pocket in an extended conformation, a 3(10) helical turn is formed by residues Leu131 to Lys134 in this segment. Unfolding of this secondary structure element can either facilitate the entry of the steroids into the binding site or modulate the important contribution that Leu131 makes to steroid binding. A comparison with previous structures supports the concept that zinc binding re-orients the side-chain of His136, and this residue serves as a lever causing disorder within the loop structure between Pro130 and Arg135

Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5{alpha}-dihydrotestosterone or C19 androgen metabolites (5{alpha}-androstan-3{beta},17{beta}-diol and 5{epsilon}{alpha}-androstan-3{beta},17{alpha}-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser42 plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp65 appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu131 and Lys134) in the loop segment (Leul31-His136) that covers the steroid-binding site as well as others (Leu171-Lys173 and Trp84) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands

Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain

Human sex hormone-binding globulin (SHBG) transports sex steroids in blood and regulates their access to target tissues. In biological fluids, SHBG exists as a homodimer and each monomer comprises two laminin G-like domains (G domains). The crystal structure of the N-terminal G domain of SHBG in complex with 5α-dihydrotestosterone at 1.55 {angstrom} resolution reveals both the architecture of the steroid-binding site and the quaternary structure of the dimer. We also show that G domains have jellyroll topology and are structurally related to pentraxin. In each SHBG monomer, the steroid intercalates into a hydrophobic pocket within the {beta}-sheet sandwich. The steroid and a 20 {angstrom} distant calcium ion are not located at the dimer interface. Instead, two separate steroid-binding pockets and calcium-binding sites exist per dimer. The structure displays intriguing disorder for loop segment Pro130-Arg135. In all other jellyroll proteins, this loop is well ordered. If modelled accordingly, it covers the steroid-binding site and could thereby regulate access of ligands to the binding pocket

Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF.

The porins OmpF and OmpC are trimeric β-barrel proteins with narrow channels running through each monomer that exclude molecules > 600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer membrane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (> 6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B(12) receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured translocation domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpF-binding sites, OBS1 (residues 2-18) and OBS2 (residues 54-63), which flank the TBE and bind with K(d)s of 2 and 24 μM, respectively, at pH 6.5 and 25 ºC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow cross-sectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a β-barrel membrane protein