Jermaine L. Jenkins

High-resolution structure of human D-glyceraldehyde-3-phosphate dehydrogenase.

GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase) is a multifunctional protein that is a target for the design of antitrypanosomatid and anti-apoptosis drugs. Here, the first high-resolution (1.75 Angstroms) structure of a human GAPDH is reported. The structure shows that the intersubunit selectivity cleft that has been leveraged in the design of antitrypanosomatid compounds is closed in human GAPDH. Modeling of an anti-trypanosomatid GAPDH inhibitor in the human GAPDH active site provides insights into the basis for the observed selectivity of this class of inhibitor. Moreover, the high-resolution data reveal a new feature of the cleft: water-mediated intersubunit hydrogen bonds that assist closure of the cleft in the human enzyme. The structure is used in a computational ligand-docking study of the small-molecule compound CGP-3466, which inhibits apoptosis by preventing nuclear accumulation of GAPDH. Plausible binding sites are identified in the adenosine pocket of the NAD(+)-binding site and in a hydrophobic channel located in the center of the tetramer near the intersection of the three molecular twofold axes. The structure is also used to build a qualitative model of the complex between GAPDH and the E3 ubiquitin ligase Siah1. The model suggests that the convex surface near GAPDH Lys227 interacts with a large shallow groove of the Siah1 dimer. These results are discussed in the context of the recently discovered NO-S-nitrosylation-GAPDH-Siah1 apoptosis cascade.

Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation.

Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ1 riboswitch from Thermoanaerobacter tengcongensis in the preQ1-bound and free states. Although the mode of preQ1 recognition is similar to that observed for preQ0, surface plasmon resonance revealed an apparent KD of 2.1 ± 0.3 nm for preQ1 but a value of 35.1 ± 6.1 nm for preQ0. This difference can be accounted for by interactions between the preQ1 methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ1-binding site, resulting in “closed” access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the “closed” free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ1 riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition.

PutA (proline utilization A) from Escherichia coli is a 1320‐amino‐acid residue protein that is both a bifunctional proline catabolic enzyme and an autogenous transcriptional repressor. Here, we report the first crystal structure of a PutA DNA‐binding domain along with functional analysis of a mutant PutA defective in DNA binding. Crystals were grown using a polypeptide corresponding to residues 1–52 of E. coli PutA (PutA52). The 2.1 Å resolution structure of PutA52 mutant Lys9Met was determined using Se‐Met MAD phasing, and the structure of native PutA52 was solved at 1.9 Å resolution using molecular replacement. Residues 3–46 form a ribbon–helix–helix (RHH) substructure, thus establishing PutA as the largest protein to contain an RHH domain. The PutA RHH domain forms the intertwined dimer with tightly packed hydrophobic core that is characteristic of the RHH family. The structures were used to examine the three‐dimensional context of residues conserved in PutA RHH domains. Homology modeling suggests that Lys9 and Thr5 contact DNA bases through the major groove, while Arg15, Thr28, and His30 may interact with the phosphate backbone. Lys9 is shown to be essential for specific recognition of put control DNA using gel shift analysis of the Lys9Met mutant of full‐length PutA. Lys9 is disordered in the PutA52 structure, which implies an induced‐fit binding mechanism in which the side chain of Lys9 becomes ordered through interaction with DNA. These results provide new insights into the structural basis of DNA recognition by PutA and reveal three‐dimensional structural details of the PutA dimer interface.

Solution Conformation and Thermodynamic Characteristics of RNA Binding by the Splicing Factor U2AF65

The U2 auxiliary factor large subunit (U2AF65) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF65 recognize polypyrimidine tract signals adjacent to 3′ splice sites. Despite the central importance of U2AF65 for splice site recognition, the relative arrangement of the U2AF65 RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF65 RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent interdomain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF65 on spliceosome components located both 5′ and 3′ to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF65 recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF65 RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF65 deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF65 RNA binding domain were compared with those of U2AF65 variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF65 to universally identify diverse pre-mRNA splice sites.

U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs

Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF65 is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3′ splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF65 recognizes degenerate Py tracts, we determined six crystal structures of human U2AF65 bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF65 bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF65 for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF65 conformations. Our results highlight both local and global conformational selection as a means for universal 3′ splice site recognition by U2AF65.

Large Favorable Enthalpy Changes Drive Specific RNA Recognition by RNA Recognition Motif Proteins

The RNA recognition motif (RRM) is a prevalent class of RNA binding domains. Although a number of RRM/RNA structures have been determined, thermodynamic analyses are relatively uncommon. Here, we use isothermal titration calorimetry to characterize single-stranded (ss)RNA binding by four representative RRM-containing proteins: (i) U2AF(65), (ii) SXL, (iii) TIA-1, and (iv) PAB. In all cases, ssRNA binding is accompanied by remarkably large favorable enthalpy changes (-30 to -60 kcal mol(-1)) and unfavorable entropy changes. Alterations of key RRM residues and binding sites indicate that under the nearly physiological conditions of these studies, large thermodynamic changes represent a signature of specific ssRNA recognition by RRMs.

An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.

A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors.

The function and localization of proteins and peptides containing C‐terminal “CaaX” (Cys‐aliphatic‐aliphatic‐anything) sequence motifs are modulated by post‐translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature‐aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off‐target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 Å resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C‐terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1‐3) are downstream of IRK and are activated by phosphatidylinositol‐dependent kinase 1 (PDK1) phosphorylating T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Aktl at S473 independent of hBVRs kinase competency. hBVR and Aktl coimmunoprecipitated, and in‐cell Förster resonance energy transfer (FRET) and glutathione S‐transferase pulldown analyses identified Aktl pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S473 independent of hBVRskinase competency. Site‐directed mutagenesis, mass spectrometry, and kinetic analyses identified S230 in hBVR 225RNRYLSF sequence as the Aktl target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR‐activated Aktl increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVRs PDK1 binding 161RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Aktl and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Aktl activation by PDKl.—Miralem, T., Lerner‐Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interactionof human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol‐dependent kinase l regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. FASEB J. 30, 2926‐2944 (20l6). www.fasebj.org