Structure of the Sgt2/Get5 complex provides insights into GET-mediated targeting of tail-anchored membrane proteins

Small, glutamine-rich, tetratricopeptide repeat protein 2 (Sgt2) is the first known port of call for many newly synthesized tail-anchored (TA) proteins released from the ribosome and destined for the GET (Guided Entry of TA proteins) pathway. This leads them to the residential membrane of the endoplasmic reticulum via an alternative to the cotranslational, signal recognition particle-dependent mechanism that their topology denies them. In yeast, the first stage of the GET pathway involves Sgt2 passing TA proteins on to the Get4/Get5 complex through a direct interaction between the N-terminal (NT) domain of Sgt2 and the ubiquitin-like (UBL) domain of Get5. Here we characterize this interaction at a molecular level by solving both a solution structure of Sgt2_NT, which adopts a unique helical fold, and a crystal structure of the Get5_UBL. Furthermore, using reciprocal chemical shift perturbation data and experimental restraints, we solve a structure of the Sgt2_NT/Get5_UBL complex, validate it via site-directed mutagenesis, and empirically determine its stoichiometry using relaxation experiments and isothermal titration calorimetry. Taken together, these data provide detailed structural information about the interaction between two key players in the coordinated delivery of TA protein substrates into the GET pathway.     

Structural polymorphism in the N-terminal oligomerization domain of NPM1

Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer–pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.Nucleophosmin (NPM1) is a highly abundant nucleolar phosphoprotein with functions associated with ribosome biogenesis (1, 2), maintenance of genome stability (1), nucleolar stress response (3), modulation of the p53 tumor suppressor pathway (4), and regulation of apoptosis (5). Importantly, genetic alterations that affect the NPM1 protein sequence or expression level are associated with oncogenesis. For example, NPM1 overexpression was observed in a variety of solid tumors, and mutations within the protein and genetic translocations involving NPM1 are associated with hematological malignancies (reviewed in ref. 6).NPM1 primarily resides in the nucleolus which is a membrane-less compartment and the site of rRNA synthesis, processing, and assembly with ribosomal proteins (7). In the nucleolus, NPM1 is involved in processing preribosomal RNA (4), chaperoning the nucleolar entry of ribosomal (1, 8) and viral (9) proteins, and stabilizing the alternate reading frame (ARF) tumor suppressor protein (4, 5, 10, 11), while also playing a role in the shuttling of preribosomal particles assembled in the nucleolus to the cytoplasm (12–14).NPM1 is a member of the nucleoplasmin protein family, which includes the histone chaperones NPM2 and NPM3. These proteins share a conserved N-terminal oligomerization domain that mediates homopentamerization (15). Disruption of NPM1 oligomerization by a small molecule (16) or an RNA aptamer (17) causes exclusive nucleoplasmic localization, loss of colocalization with ARF, and induction of p53-dependent apoptosis (16, 17). These observations suggest that changes in the oligomeric state of NPM1 may influence its biological functions. However, although it is hypothesized (1) that NPM1 function is modulated through control of its oligomeric state, experimental data are currently lacking. Intriguingly, NPM1 exhibits 40 putative phosphorylation sites, the majority of which are evolutionarily conserved (18, 19). Modification of these sites that is influenced by subcellular localization and cell cycle phase (20, 21) modulates the biological function of NPM1 (1, 6). Approximately one third of these phosphorylation sites are located within the N-terminal oligomerization domain, indicating their possible involvement in regulation of the oligomerization state of NPM1.What is the molecular mechanism that underlies the various functions of NPM1? Here we show by using in vitro biophysical and structural methods that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating a range of conformations with various degrees of structural disorder that span two extreme structural states: a folded pentameric state and a disordered monomeric state. Several conserved phosphorylation sites in pentameric Npm-N are positioned within the hydrophobic interior of the pentameric structure (18) and therefore are inaccessible to kinases. Interestingly, other conserved sites are solvent accessible, and we show that these posttranslational modification (PTM) sites serve as molecular switches for modulating the oligomerization and the folding state of Npm-N. We propose that, when exposed through initial destabilizing phosphorylation events, the otherwise inaccessible sites act as molecular locks that, when phosphorylated (22–24), destabilize the pentameric form and lock Npm-N in the monomeric state. We demonstrate that the monomer–pentamer equilibrium is modulated by protein binding partners and have identified a short, arginine-rich (R-rich) linear motif that mediates this interaction. Our results suggest that the diverse cellular functions and subcellular localization of NPM1 are influenced through a regulated unfolding mechanism, signaled through PTMs and intermolecular interactions.     

Structure of a eukaryotic thiaminase I

Thiaminases, enzymes that cleave vitamin B1, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes catalyze the elimination of the thiazole ring moiety from thiamin through substitution of the methylene group with a nitrogenous base or sulfhydryl compound. In eukaryotic organisms, these enzymes are reported to have much higher molecular weights than their bacterial counterparts. A thiaminase I of the single-celled amoeboflagellate Naegleria gruberi is the only eukaryotic thiaminase I to have been cloned, sequenced, and expressed. Here, we present the crystal structure of N. gruberi thiaminase I to a resolution of 2.8 Å, solved by isomorphous replacement and pseudo–two-wavelength multiwavelength anomalous diffraction and refined to an R factor of 0.231 (R_free, 0.265). This structure was used to solve the structure of the enzyme in complex with 3-deazathiamin, a noncleavable thiamin analog and enzyme inhibitor (2.7 Å; R, 0.233; R_free, 0.267). These structures define the mode of thiamin binding to this class of thiaminases and indicate the involvement of Asp272 as the catalytic base. This enzyme is able to use thiamin as a substrate and is active with amines such as aniline and veratrylamine as well as sulfhydryl compounds such as l-cysteine and β-mercaptoethanol as cosubstrates. Despite significant differences in polypeptide sequence and length, we have shown that the N. gruberi thiaminase I is homologous in structure and activity to a previously characterized bacterial thiaminase I.The nonpathogenic unicellular protozoan Naegleria gruberi is a ubiquitous eukaryote, found in aerobic and microaerobic environments including freshwater, freshwater silt, and moist soils worldwide (1–4). It exists predominantly as an amoeba but is able to undergo a rapid phenotypic change into a streamlined swimming flagellate when subjected to a nutrient-poor environment (3). Naegleria is also able to form resting cysts, which are then capable of excysting back to amoebae (2).Extracts of N. gruberi have been shown to possess a proteinaceous agent that is “cytopathogenic” to vertebrate cells (5). Fulton and Lai show that these extracts of N. gruberi amoebae cause generations-delayed apoptotic death of both proliferating and quiescent vertebrate cells in culture (6). The apoptosis-inducing agent was isolated based on activity, and characterized and identified as a thiaminase I, an enzyme that degrades thiamin (vitamin B1) and thiamin diphosphate (TPP), the biologically active form of thiamin (7). They showed that N. gruberi thiaminase I induces apoptosis via its enzymatic activity in that an enzymatically inactive mutant lost its ability to cause cell death (7). Active thiaminase I depletes extracellular thiamin, which leads to subsequent depletion of intracellular TPP, an essential coenzyme for many enzymes involved in carbohydrate and energy metabolism, and in turn triggers apoptosis by an as-yet-to-be-characterized mechanism. It has long been known that thiamin deficiency in animals can cause neurological and cardiac symptoms that ultimately lead to death of the animal. In humans, beriberi and Wernicke–Korsakoff syndrome are associated with chronic thiamin deficiency (8). Fulton and Lai have shown that the N. gruberi thiaminase I is also capable of killing cells resistant to commonly used chemotherapeutic agents and suggest that thiamin depletion-induced cell death may be a suitable candidate for use in tissue-targeted cancer therapies (6).Thiaminases catalyze the cleavage of biologically active thiamin into its pyrimidine and thiazole ring moieties (9, 10). These enzymes can be grouped into two classes defined by the nucleophile used in the mechanism by which the cleavage is accomplished. The thiaminase II (EC 3.5.99.2) class of enzymes exclusively uses water to accelerate the hydrolysis of thiamin into 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole and is found only in bacteria, fungi, and yeast (11). The thiaminase I (EC 2.5.1.2) class of enzymes uses a variety of aromatic and heterocyclic amines and sulfhydryl compounds as substituting bases in a nucleophilic displacement reaction on the methylene group of the pyrimidine moiety (10, 12–15). Thiaminase I is found in specific species of microorganisms such as Bacillus and Clostridium as well as multicellular organisms including certain ferns, insects, shellfish, and freshwater and ocean fish (10, 14, 16–26). Although distributed throughout the kingdoms, the phylogenetic distribution of thiaminase I, unlike most other enzymes, appears to follow no easily discernable evolutionary pattern. A physiological role has yet to be assigned for thiaminase I (27), and several studies report that thiaminase I enzymes from eukaryotic organisms are produced as larger holoenzymes, 55–200 kDa, some of which are active as smaller fragments (17, 21–23, 25, 26). The presence of this enzyme in the diet of animals can have several deleterious effects and can even be fatal. It is responsible for thiamin deficiency and early mortality syndrome in Great Lakes salmonines, polioencephalomalacia in sheep, and cerebrocortical necrosis in cattle (9, 28, 29). Consumption of silk worm larvae as a source of protein in Nigeria causes acute seasonal cerebellar ataxia (22). Finally, the 1860–1861 expedition of Burke and Wills across Australia turned fatal when the men, whose diet consisted primarily of raw nardoo fern, suffered from thiaminase poisoning, developed beriberi, and died (30). It is tempting to speculate that these organisms have acquired thiaminase I through horizontal gene transfer and maintained it in their genomes as a mechanism of defense.The N. gruberi thiaminase I is part of a large polypeptide sequence of ∼1,025 aa residues (D2V4Z5; Fig. 1). The C-terminal portion of this sequence (669 aa) is homologous to transketolases. It is 49% identical in sequence to the Saccharomyces cerevisiae transketolase ({"type":"entrez-protein","attrs":{"text":"P23254","term_id":"1351256","term_text":"P23254"}}P23254) and includes conservation of key residues involved in dimer stabilization, cofactor binding, and catalysis in the yeast enzyme. Attempts to isolate the full-length protein from extracts of N. gruberi amebae have been unsuccessful; however, the thiaminase I activity survives, indicating that the thiaminase I domain of the protein remains intact. In addition, the N-terminal domain expressed by itself (356 aa) has thiaminase I activity in vitro that is comparable to the activity of the thiaminase I domain isolated from cellular extracts (6).Open in a separate windowFig. 1.The gene that encodes the thiaminase I examined in this study yields a protein with homology to both thiaminase I and transketolase enzymes. Key active-site residues are indicated.Given the low sequence identity (∼25%) of the thiaminase I isolated from N. gruberi (Ng-thiaminase) to the previously characterized Bacillus thiaminolyticus (Paenibacillus thiaminolyticus) thiaminase I ({"type":"entrez-protein","attrs":{"text":"P45741","term_id":"1174670","term_text":"P45741"}}P45741) (Bt-thiaminase) and the fact that the N. gruberi enzyme is part of a larger protein, we endeavored to determine its structure. Additionally, the sequence of the Bt-thiaminase enzyme contains only one cysteine, which is located in the active site, whereas the sequence of the Ng-thiaminase enzyme possesses six cysteines. The structure of the Bt-thiaminase has been solved bound to a mechanism-based inhibitor (31), 4-amino-6-chloro-2,5-dimethylpyrimidine (Pyd) (Fig. S1); however, this molecule represents only the pyrimidine portion of thiamin and may not be representative of the mode of substrate binding to this class of thiaminases. Here, we describe the X-ray crystal structures of a thiaminase I from N. gruberi in its unliganded state and bound to a substrate analog, 3-deazathiamin (3-dzThi) (Fig. S1), which is isoelectronic with and essentially identical in shape and size to thiamin (32, 33). The N. gruberi thiaminase I has been shown to use thiamin, but not thiamin diphosphate, as a substrate and is active with both amines and sulfhydryl compounds as cosubstrates. Despite low sequence identity and differences in native polypeptide length, the tertiary structure of the thiaminase I from Naegleria gruberi closely resembles that of thiaminase I from Bacillus thiaminolyticus.     

Structure of an intermediate conformer of the spindle checkpoint protein Mad2

The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.Accurate chromosome segregation requires proper, dynamic attachment of sister chromatids to spindle microtubules during mitosis, which enables chromosome alignment at the metaphase plate (1, 2). At metaphase, two opposing kinetochores of a sister-chromatid pair attach to microtubules emanating from opposite spindle poles. This bipolar kinetochore–microtubule attachment enables all sister chromatids to align at the metaphase plate. The anaphase-promoting complex or cyclosome (APC/C), along with its activator cell division cycle 20 (Cdc20), indirectly activates the protease separase through triggering the ubiquitination and degradation of the separase inhibitors, securin and cyclin B1 (3–5). Active separase then cleaves cohesin, leading to chromosome segregation (6, 7). The separated chromatids are evenly partitioned into the two daughter cells through their attachment to the spindle.During mitotic progression, not all sister kinetochores achieve bipolar attachment synchronously. The spindle checkpoint senses the existence of kinetochores not attached or improperly attached to spindle microtubules and inhibits APC/C^Cdc20 through promoting the formation of the APC/C-inhibitory mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, mitotic arrest deficient 2 (Mad2), and Cdc20 (8–12). APC/C inhibition delays separase activation, cohesin cleavage, and the onset of chromosome segregation and provides time for unattached kinetochores to reach proper attachment before separation. The spindle checkpoint thus ensures the fidelity of chromosome segregation.The unusual multistate behavior of the checkpoint protein Mad2 is critical for kinetochore-dependent spindle checkpoint signaling (9, 12–14). Mad2 has multiple folded conformers, including the latent, open conformer (O-Mad2) and the activated, closed conformer (C-Mad2) (15–19). C-Mad2 binds to its upstream regulatory protein Mad1 and its downstream target Cdc20 through the Mad2-interacting motif (MIM) (4, 16, 17). C-Mad2 topologically entraps this MIM motif through a seat-belt-like structure formed by its C-terminal region (16, 17). O-Mad2 cannot interact with Mad1 or Cdc20 because the seat belt is not formed in O-Mad2 and the C-terminal region in O-Mad2 blocks the ligand-binding site (16). In human cells, Mad2 exists as multiple species, including free, latent O-Mad2 and C-Mad2 tightly bound to Mad1 (18, 20). Upon checkpoint activation, the Mad1–Mad2 core complex is targeted to unattached kinetochores (21, 22). This core complex can further recruit additional copies of O-Mad2 and convert them into intermediates termed I-Mad2 (Fig. 1A) (19, 23–25). Cdc20 and BubR1–Bub3 are also recruited to unattached kinetochores through other mechanisms (26, 27). The close proximity of I-Mad2 and Cdc20 stimulates efficient formation of the C-Mad2–Cdc20 complex, which can then associate with BubR1–Bub3 to form MCC (28). Thus, unattached kinetochores promote the conformational activation of Mad2 and the production of MCC.Open in a separate windowFig. 1.Crystal structure of the asymmetric I-Mad2–C-Mad2 dimer. (A) Model for conformational activation of Mad2 at kinetochores during mitosis. MCC, mitotic checkpoint complex; MIM, Mad2-interacting motif. (B) Diagram of the crystal structure of the asymmetric Mad2^ΔN dimer, with the I-Mad2 and C-Mad2 monomers colored purple and blue, respectively. The entrapped C-terminal tail (C-tail) of another Mad2^ΔN molecule through crystal packing interactions is shown and colored orange. (C) Superimposed diagrams of the C-Mad2 monomer in the Mad2^ΔN dimer (blue) and the C-Mad2 monomer in the symmetric Mad2^L13A dimer (green). The entrapped N-terminal region (NR) of another Mad2^L13A molecule through crystal packing interactions is shown and colored red. The C- and N-terminal regions in Mad2^L13A that underwent large conformational changes from O-Mad2 to C-Mad2 are colored yellow. (D) Superimposed diagrams of the I-Mad2 monomer in the Mad2^ΔN dimer (purple) and the solution structure of O-Mad2^ΔNC lacking both the N- and C-terminal 10 residues (cyan). All structural figures were generated with PyMol (https://www.pymol.org).Previous NMR analyses showed that the chemical shifts of many backbone amides and methyl groups in I-Mad2 were drastically different from those in O-Mad2 or C-Mad2 (23, 24). These results seemingly supported the notion that I-Mad2 adopted a fold different from those of O- or C-Mad2. It was further suggested that I-Mad2 might have undergone partial unfolding in the N- and C-terminal regions, which was a prerequisite for the topological entrapment of Cdc20 and formation of C-Mad2.In a breakthrough study, Musacchio and coworkers determined the structure of an asymmetric Mad2 dimer (29). In this dimer, one monomer was C-Mad2 bound to an unnatural peptide ligand called Mad2-binding peptide 1 (MBP1). The other monomer was a Mad2 mutant with a shortened loop (termed loop-less or LL), which was thought to block the formation of I-Mad2. Indeed, in this asymmetric dimer, Mad2^LL had the same overall fold as O-Mad2. This dimer was thus proposed to represent the docking complex of O-Mad2–C-Mad2 (Fig. 1A) (29).To gain structural insights into the conformational activation of Mad2, we determined the crystal structure of the I-Mad2–C-Mad2 dimer and analyzed its conformation in solution with NMR. Our studies show that, similar to Mad2^LL, I-Mad2 does not undergo partial unfolding and retains the same fold as O-Mad2, both in crystal and in solution. Instead, the dramatic chemical shift changes of I-Mad2 are likely caused by the relative rotations between the dimerization and central helices, as well as the rigid body shift of the β hairpin that contacts both helices. These rearrangements render the I-Mad2 core more closely resemble the C-Mad2 core. Our finding that I-Mad2 remains folded at the C-terminal region suggests that the partially unfolded Mad2 species ready for Cdc20 entrapment likely represents a fleeting transition state, not a populated intermediate as widely believed. Furthermore, mutations of I-Mad2 residues at or near the dimerization interface reduce the extent of C-Mad2–induced conformational rearrangements and impede the spontaneous O-C Mad2 structural transition. Our studies thus establish the structural basis for the allosteric communication between the dimerization interface and the C-terminal malleable region of Mad2.     

DNA polymorphisms of the KiSS1 3' untranslated region interfere with the folding of a G-rich sequence into G-quadruplex

KISS1R and its ligand, the kisspeptins, are key hypothalamic factors that regulate GnRH hypothalamic secretion and therefore the pubertal timing. During studies analysing KiSS1 as a candidate gene in pubertal onset disorders, two SNP and one nucleotide insertion were observed in a 23 nucleotides G-rich sequence located 65 nucleotides downstream of the stop codon. The polymorphisms formed four haplotypes. Biophysical experiments revealed the ability of this G-rich sequence to fold into G-quadruplex structures and demonstrated that the three DNA polymorphisms did not perturb the folding into G-quadruplex but affected G-quadruplex conformation. A functional luciferase reporter-based assay revealed functional differences between 3'UTR haplotypes. These data show that polymorphisms in a G-rich sequence of the 3'UTR of KISS1, able to fold into G-quadruplex structures, can modulate gene expression. They highlight the potential role of this G-quadruplex in the regulation of KISS1 expression and in the timing of pubertal onset.     

Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI

IsdI, a heme-degrading protein from Staphylococcus aureus, binds heme in a manner that distorts the normally planar heme prosthetic group to an extent greater than that observed so far for any other heme-binding protein. To understand better the relationship between this distinct structural characteristic and the functional properties of IsdI, spectroscopic, electrochemical, and crystallographic results are reported that provide evidence that this heme ruffling is essential to the catalytic activity of the protein and eliminates the need for the water cluster in the distal heme pocket that is essential for the activity of classical heme oxygenases. The lack of heme orientational disorder in (1)H-NMR spectra of the protein argues that the catalytic formation of β- and δ-biliverdin in nearly equal yield results from the ability of the protein to attack opposite sides of the heme ring rather than from binding of the heme substrate in two alternative orientations.     

Molecular mechanisms for the regulation of histone mRNA stem-loop–binding protein by phosphorylation

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.Histone synthesis increases at the beginning of S-phase to package newly replicated DNA with histone proteins, but synthesis must be shut down rapidly and histone mRNA degraded at the end of DNA replication because of the toxicity of surplus histone proteins (1, 2). This cyclic demand for histones requires strict regulation, which is achieved mainly by controlling the synthesis and degradation of histone mRNA (3). Replication-dependent histone mRNAs are the only known cellular mRNAs that are not polyadenylated and instead end with a conserved stem loop (4). Histone mRNAs are generated from longer histone pre-mRNAs as a result of an endonucleolytic cleavage between the stem loop and a purine-rich downstream sequence termed the “histone downstream element” (HDE) (5).Stem-loop–binding protein (SLBP), also known as “hairpin-binding protein” (6), binds to the histone mRNA stem loop, and U7 small nuclear ribonucleoprotein binds to the HDE (7). Other factors, including the endonuclease CPSF-73, are involved in both polyadenylation and histone mRNA 3′-end processing (8–11). In mammalian nuclear extracts, SLBP is not absolutely required for the biochemical reaction of processing (12). In contrast, cleavage of histone pre-mRNA in Drosophila cells and nuclear extracts requires the binding of SLBP to the stem loop (10, 13).The minimal histone mRNA processing domain of Drosophila SLBP contains a 72-aa RNA-binding domain (RBD) unique to SLBPs and an 18-aa C-terminal region (Fig. 1A) (14). This RNA-processing domain (RPD) is necessary and sufficient for histone mRNA 3′-end processing in vitro (15). The RBDs of human SLBP (hSLBP) and Drosophila SLBP (dSLBP) are phosphorylated at a Thr residue in a conserved TPNK motif (16, 17). The recent crystal structure of hSLBP RBD in complex with histone mRNA stem loop and 3′ hExo, a 3′–5′ exonuclease required for histone mRNA degradation, provided the first molecular insights into the architecture of this complex, and revealed how the hSLBP RBD forms a new RNA-binding motif to interact with the stem-loop RNA (18). On the other hand, how SLBP alone interacts with the RNA or how this interaction might be affected by phosphorylation of the TPNK motif is not known.Open in a separate windowFig. 1.Schematic of the domain architecture of dSLBP (Upper) and amino acid sequence alignment of RPDs of Drosophila and human SLBP (Lower). Domains of SLBP include the N-terminal domain (NTD), RBD, and C-terminal region (C). Amino acid sequences are shown with the RBD sequence in the top two rows and the C-terminal region in the bottom row. T230 in the TPNK motif and phosphorylation sites in the C-terminal region are indicated with boldface and asterisks, respectively; the residues involved in RNA binding are shown in cyan; and acidic residues in the C-terminal region are shown in red.The C-terminal region of dSLBP contains a motif, SNSDSDSD, whose hyperphosphorylation is required for efficient processing of histone pre-mRNA (15). Despite the similarity of hSLBP and dSLBP RBDs (55% identical residues) and their ability to bind identical stem-loop RNA sequences, neither SLBP can substitute for the other to process histone pre-mRNA in nuclear extracts; in fact, hSLBP inhibits processing of Drosophila histone pre-mRNA (15). This incompatibility results from differences in the C-terminal region (Fig. 1). The sequence C-terminal to the RBD in hSLBP is required for processing, but it is longer, has no similarity to the Drosophila sequence, and lacks phosphorylation sites.Here we focused on dSLBP and showed that phosphorylation greatly increases dSLBP binding affinity for the histone mRNA stem loop. Mimicking phosphorylation of the dSLBP RPD by mutation of phosphorylation sites to Glu residues at both the TPNK motif and the C-terminal region also boosted binding affinity relative to the nonphosphorylated dSLBP RPD. Structural studies of both the human and Drosophila SLBP RPD indicated that phosphorylation of the TPNK motif stabilizes the RNA-binding domain, but the C-terminal region is flexible in the protein:RNA complex and does not contact the RNA. Instead, we show that the increased negative charge in the C-terminal region of the dSLBP RPD results in a more compact ensemble of protein conformations in the absence of RNA, thereby increasing RNA-binding affinity by reducing the entropy of the unbound protein.     

Structure of a bacterial toxin-activating acyltransferase

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host–cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.Pathogenic bacteria secrete pore-forming protein toxins (PFTs) that target tissue and immune cell membranes to aid colonization and survival during infections, subvert cell signaling, induce apoptosis, and promote cell lysis (1–8). Among Gram-negative bacteria, large PFTs are secreted by pathogenic species of Pasteurella, Actinobacillus, Proteus, Morganella, Moraxella, and Bordetella, exemplified by the 110-kDa hemolysin (HlyA) of uropathogenic and enterohemorrhagic Escherichia coli. These toxins play important roles in cystitis and pyelonephritis, hemorrhagic intestinal disease, periodontitis, pneumonia, septicemia, whooping cough, and wound infections (4), and unusually they are made as an inactive protoxin, requiring posttranslational activation before export (9–12).Reconstituting the toxin activation reaction in vitro some time ago demonstrated that the essential modification is a novel fatty acid acylation, affected by a specialized coexpressed toxin-activating acyltransferase, in E. coli HlyC, that uses acyl-acyl carrier protein (acyl-ACP) as the fatty acid donor (4, 13, 14). The acyltransferase does not share significant sequence identity with other bacterial and eukaryotic enzymes, and cellular acyltransferases from either the host or pathogen cannot substitute for HlyC in toxin activation. HlyC independently binds two separate 50- to 80-aa transferase recognition domains (15), each encompassing one of the internal target lysines K564 and K690 of E. coli protoxin HlyA, which are acylated by amide linkage, heterogeneously with fatty acids containing 14, 15, and 17 carbon chains (16, 17). Loss of the HlyC binding domain or substitution of protoxin K564 and K690 prevents fatty acyl modification and abrogates all toxin activity (14) as does loss of the transferase (18).Acylation is essential to the entire family of pore-forming toxins, Bordetella pertussis proCyaA lysine acylation has also been demonstrated (19), and the toxin-activating acyltransferases (which we now call TAATs) have high sequence similarity and cross-activate other protoxins (4, 20–22). The TAAT activation mechanism is seemingly unique, and extensive site-directed mutagenesis has so far only identified a single potentially catalytic residue, His23 of HlyC (23–25). Structural information is essential to understand the toxin activation mechanism and assess TAATs as a potential target for developing antivirulence compounds that do not affect the host commensal flora. Here, we determine the TAAT crystal structure, solution state, and likely active site.     

Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli beta-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with beta and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to beta. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with beta, and that the beta-clamp may represent an antibiotic target.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein

The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a ∼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2−specific sequence, ₇₃YIDI₇₆. Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.

The severity of the current COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relative to past outbreaks of Middle East respiratory syndrome, SARS, and other betacoronaviruses in humans begs the question as to its molecular basis. The accessory protein ORF8 is one of the most rapidly evolving betacoronavirus proteins (1–7). While ORF8 expression is not strictly essential for SARS-CoV and SARS-CoV-2 replication, a 29-nucleotide deletion (Δ29) that occurred early in human to human transmission of SARS-CoV, splitting ORF8 into ORF8a and ORF8b, is correlated with milder disease (8). A 382-nucleotide deletion (Δ382) in SARS-CoV-2 (9, 10) was also found to correlate with milder disease and a lower incidence of hypoxia (11).SARS-CoV-2 ORF8 is a 121-amino acid (aa) protein consisting of an N-terminal signal sequence followed by a predicted Ig-like fold (12). With <20% sequence identity to SARS-CoV ORF8, SARS-CoV-2 ORF8 is remarkably divergent. ORF8 proteins from both viruses possess a signal sequence for endoplasmic reticulum (ER) import. Within the lumen of the ER, SARS-CoV-2 ORF8 interacts with a variety of host proteins, including many factors involved in ER-associated degradation (13). Presumably, ORF8 is secreted, rather than retained in the ER, since ORF8 antibodies are one of the principal markers of SARS-CoV-2 infections (14). Several functions have been proposed for SARS-CoV-2 ORF8. ORF8 disrupts IFN-I signaling when exogenously overexpressed in cells (15). It has been shown that ORF8 of SARS-CoV-2, but not ORF8 or ORF8a/ORF8b of SARS-CoV, down-regulates MHC-I in cells (16).These observations suggest the relationship between ORF8 structure, function, and sequence variation may be pivotal for understanding the emergence of SARS-CoV-2 as a deadly human pathogen. Yet not only is there no three-dimensional structure of any ORF8 protein from any coronavirus, there are no homologs of known structure with sequence identity sufficient for a reliable alignment. SARS and SARS-CoV-2 ORF7a are the most closely related templates of known structure (17), yet their core is approximately half the size of ORF8, and their primary sequence identity is negligible. Therefore, we determined the crystal structure of SARS-CoV-2 ORF8. The structure confirms the expected Ig-like fold and overall similarity of the core fold to SARS-CoV-2 ORF7a. The structure reveals two novel dimer interfaces for SARS-CoV-2 ORF8 unique relative to all but its most recent ancestors in bats. Together, our results set the foundation for elucidating essential aspects of ORF8 biology to be leveraged for the development of novel therapeutics.     

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.Aptamers are artificially selected oligonucleotides that bind to molecular targets, typically proteins, with high specificity and avidity (1–3). DNA aptamers have been selected against dozens of targets for biomedical applications both as therapeutics (4, 5) and diagnostics (6, 7). Despite their widespread application, few DNA aptamer-target complex structures have been solved (8)–the best studied of which is the G-quadruplex aptamer that binds to thrombin (9–12). A DNA aptamer that binds to von Willebrand factor showed a three-stem structure of mainly B-form DNA with some noncanonical base pairing (13). Most recently, the structure of an innovative Slow Off-rate Modified Aptamer (SOMAmer) bound to platelet-derived growth factor B was solved, revealing binding via a hydrophobic surface that mimics how the factor binds to its receptor (14). Generally, the lack of DNA aptamer-target structures has limited our understanding of the mechanisms by which DNA aptamers attain their specificity (15), resulting in a bias in aptasensor development (16).Better point-of-care tests are critically needed for malaria, a disease which continues to claim more than 1 million lives globally every year (17). Antimalarial drugs have been administered presumptively to patients with fever for decades, leading to drug resistance and poor management of other febrile illness. The cost of newer, more effective treatments has led to a situation whereby improved diagnostics has become a major factor that could reduce the burden of malaria in the developing world (17). Antibody-based rapid diagnostic tests have greatly benefitted malaria management, but significant issues with cost (17) and stability in tropical climates (18) remain that are intrinsically associated with the use of protein antibodies. DNA aptamers compare favorably to antibodies for diagnostic applications (19) with particular advantages that could be critical for diagnostic tests of the developing world: thermal stability, convenient chemical synthesis, and potentially lower costs of production (16). Here, we report the crystal structure and application of a unique DNA aptamer against an established malaria pan-species diagnostic target, Plasmodium falciparum lactate dehydrogenase (PfLDH) (20), and a mechanism of molecular recognition by a distorted hairpin DNA aptamer.     

Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair

UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A^DDB2) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization. The functional significance of the dimeric UV-DDB [(DDB1-DDB2)₂], in a complex with damaged DNA, is validated by electron microscopy, atomic force microscopy, solution biophysical, and functional analyses. We propose that the binding of UV-damaged DNA results in conformational changes in the N-terminal domain of DDB2, inducing helical folding in the context of the bound DNA and inducing dimerization as a function of nucleotide binding. The temporal and spatial interplay between domain ordering and dimerization provides an elegant molecular rationale for the unprecedented binding affinities and selectivities exhibited by UV-DDB for UV-damaged DNA. Modeling the DDB1-CUL4A^DDB2 complex according to the dimeric UV-DDB-AP24 architecture results in a mechanistically consistent alignment of the E3 ligase bound to a nucleosome harboring damaged DNA. Our findings provide unique structural and conformational insights into the molecular architecture of the DDB1-CUL4A^DDB2 E3 ligase, with significant implications for the regulation and overall organization of the proteins responsible for initiation of NER in the context of chromatin and for the consequent maintenance of genomic integrity.     

Structural biology of Mycobacterium tuberculosis proteins: the Indian efforts

Among the many different objectives of large scale structural genomics projects are expanding the protein fold space, enhancing understanding of a model or disease-related organism, and providing foundations for structure-based drug discovery. Systematic analysis of protein structures of Mycobacterium tuberculosis has been ongoing towards meeting some of these objectives. Indian participation in these efforts has been enthusiastic and substantial. The proteins of M. tuberculosis chosen for structural analysis by the Indian groups span almost all the functional categories. The structures determined by the Indian groups have led to significant improvement in the biochemical knowledge on these proteins and consequently have started providing useful insights into the biology of M. tuberculosis. Moreover, these structures form starting points for inhibitor design studies, early results of which are encouraging. The progress made by Indian structural biologists in determining structures of M. tuberculosis proteins is highlighted in this review.     

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.     

Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release

We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 Å resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2′-endo conformation, and the 2′ hydroxyl of A76 is within hydrogen-bond distance of the 2′ hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.     

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in (ProProGly)₇ collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T_m values that are increased by > 50 °C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 Å reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2/3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.     

Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans

Candida albicans is the most prevalent fungal pathogen in humans and a major source of life-threatening nosocomial infections. The Als (agglutinin-like sequence) glycoproteins are an important virulence factor for this fungus and have been associated with binding of host-cell surface proteins and small peptides of random sequence, the formation of biofilms and amyloid fibers. High-resolution structures of N-terminal Als adhesins (NT-Als; up to 314 amino acids) show that ligand recognition relies on a motif capable of binding flexible C termini of peptides in extended conformation. Central to this mechanism is an invariant lysine that recognizes the C-terminal carboxylate of ligands at the end of a deep-binding cavity. In addition to several protein-peptide interactions, a network of water molecules runs parallel to one side of the ligand and contributes to the recognition of diverse peptide sequences. These data establish NT-Als adhesins as a separate family of peptide-binding proteins and an unexpected adhesion system for primary, widespread protein-protein interactions at the Candida/host-cell interface.     

The Shr receptor from Streptococcus pyogenes uses a cap and release mechanism to acquire heme–iron from human hemoglobin

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a “cap and release” mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr–Hb complex which function to transiently uncap HID1 from the heme bound to Hb’s β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr’s NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

To successfully mount infections bacterial pathogens must overcome host nutritional immunity mechanisms that limit access to iron, an essential metal nutrient required for microbial survival because it functions as a cofactor in enzymes that mediate cellular metabolism. Human hemoglobin (Hb) contains ~75 to 80% of the body’s total iron in the form of heme (iron–protoporphyrin IX) and is thus a prime nutrient source for invading microbes (1–9). Bacteria gain access to Hb’s iron-laden heme molecules when erythrocytes are ruptured by bacterial cytotoxins or when they spontaneously lyse. In gram-positive monoderm bacteria, extracellular Hb is captured by surface-displayed microbial receptors. Hb’s heme molecules are then released and transferred via microbial heme-binding chaperones across the expanse of the peptidoglycan to the membrane, where they are imported into the cell and degraded to release iron. The acquisition mechanisms that many pathogens use to bind to Hb and remove its tightly bound heme molecules are not well understood. Streptococcus pyogenes (group A Streptococcus) colonizes the skin and mucosal surfaces in humans and is estimated to cause more than 500,000 deaths annually (10–12). It causes a range of illnesses, ranging from acute pharyngitis to life-threatening diseases such as scarlet fever, bacteremia, pneumonia, necrotizing fasciitis, myonecrosis, and streptococcal toxic shock syndrome (13, 14). S. pyogenes employs the streptococcal hemoprotein receptor (Shr) to capture Hb and acquire its heme molecules, and it is an important virulence factor that when genetically deleted reduces the ability of the pathogen to grow in human blood and to cause infections in murine and zebrafish models (15–17). Strategies that interfere with the ability of S. pyogenes and other pathogenic bacteria to harvest heme from Hb could be useful in treating infections, as they would effectively starve pathogens of iron.The S. pyogenes Shr protein is a structurally unique multidomain Hb receptor that is also found in other streptococci and clostridia species (e.g., Clostridium novyi, Streptococcus iniae, Streptococcus equi, and Streptococcus dysgalactiae) (Fig. 1A). Its N-terminal region (NTR, residues 26 to 364) binds to Hb using two Hb interacting domains (HIDs), called HID1 and HID2 (formally known as DUF1533 domains) (18, 19). The HIDs are structurally novel binding modules and are joined via a structured linker domain (L) to a C-terminal region (CTR, residues 365 to 1,275) which contains two heme-binding NEAr iron Transporter domains (NEAT domains N1 and N2) that are separated by a series of leucine-rich repeats (LRR). The NTR and N1 domain within Shr (called NTR-N1) preferentially bind to holo-Hb and remove its heme (18). In vitro, heme bound by the N1 domain is then readily transferred to either the C-terminal N2 domain, or to Shp, a cell wall-associated protein that relays heme to the membrane-associated HtsABC/SiaABC transporter that pumps heme into the cytoplasm (20–22). The N2 domain in Shr may act as a storage unit, since it binds to heme with much higher affinity than N1 and does not directly transfer heme to Shp (23). Shr also interacts via its N2 domain with the human extracellular matrix (ECM) proteins fibronectin and laminin (15, 16, 18), and its exposure on the cell surface may make it a useful epitope in S. pyogenes vaccines (24, 25). However, it remains poorly understood how Shr acquires heme from Hb. Here we show using a combination of biophysical and structural methods that Shr uses its HIDs to selectively bind to the heme-loaded form of Hb, slowing the rate of heme release by directly contacting the edges of its protoporphyrin rings. However, receptor dynamics within the Shr–Hb complex act to transiently uncap the HIDs from Hb’s β subunit, enabling heme’s gated release and subsequent capture by the receptor. This “cap and release” mechanism exploits the β subunit’s inherent weaker affinity for heme (26), allowing S. pyogenes to preferentially capture only heme-saturated forms of Hb that contain iron.Open in a separate windowFig. 1.Structure of the Hb–Shr^H2 complex. (A) Domain schematic of the Shr receptor. The polypeptide constructs used in this study are shown below. (B) Crystal structure of the Hb–Shr^H2 complex. The asymmetric unit of the crystal contains two tetramers of Hb that are bound by three molecules of Shr^H2. (C and D) HID2 binds over the heme pockets in both the α and β chains of Hb. These capping interactions directly contact both the heme and globin chain, burying an average of ~153 Ų and ~408 Ų of solvent-accessible surface area, respectively. Hb contacts originate from three surface loops in HID2: β2-α1, β4-β5, and β5-β6. (E) Expanded view of the Hb-receptor interface showing interactions with the heme molecule bound to the α subunit. The heme molecules are shown in stick format with oxygen and nitrogen atoms colored red and blue, respectively. Side chains in the receptor that interact with Hb are shown in stick format. (F) Identical to panel (E), except that receptor contacts to the β subunit in Hb are shown. Hb is in its ferric form. Color scheme: α subunit (salmon), β subunit (green), and HID2 (blue).     

Ionic strength-dependent persistence lengths of single-stranded RNA and DNA

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (l(p)) of ssRNA. We observe valence and ionic strength-dependent differences in l(p) in a direct comparison between 40-mers of deoxythymidylate (dT(40)) and uridylate (rU(40)) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.