Krasimir Spasov - Academia.edu (original) (raw)

Papers by Krasimir Spasov

Research paper thumbnail of DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function

Molecular and cellular biology, 2015

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs... more DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, i...

Research paper thumbnail of Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase

Bioorganic & Medicinal Chemistry Letters, 2015

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a... more Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

Research paper thumbnail of DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function

Molecular and cellular biology, 2015

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs... more DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, i...

Research paper thumbnail of Structure-Based Evaluation of Non-nucleoside Inhibitors with Improved Potency and Solubility That Target HIV Reverse Transcriptase Variants

Journal of medicinal chemistry, Jan 5, 2015

The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV... more The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of comp...

Research paper thumbnail of Illuminating HIV gp120-ligand recognition through computationally-driven optimization of antibody-recruiting molecules

Chemical Science, 2014

Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV... more Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV gp120 (ARM-H). These studies have leveraged a combination of medicinal chemistry, biochemical and cellular assay analysis, and computation. Our findings have afforded an optimized analog of ARM-H, which is ~1000 fold more potent in gp120-binding and MT-2 antiviral assays than our previously reported derivative. Furthermore, computational analysis, taken together with experimental data, provides evidence that azaindole- and indole-based attachment inhibitors bind gp120 at an accessory hydrophobic pocket beneath the CD4-binding site and can also adopt multiple unique binding modes in interacting with gp120. These results are likely to prove highly enabling in the development of novel HIV attachment inhibitors, and more broadly, they suggest novel applications for ARMs as probes of conformationally flexible systems.

Research paper thumbnail of RAC Protein Directs the Complete Removal of the 3′ External Transcribed Spacer by the Pac1 Nuclease

Molecular Cell, 2002

In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein c... more In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS. Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence. The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.

Research paper thumbnail of Picomolar Inhibitors of HIV-1 Reverse Transcriptase: Design and Crystallography of Naphthyl Phenyl Ethers

ACS Medicinal Chemistry Letters, 2014

Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nuc... more Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Promising compounds are reported that show midpicomolar activity against the wild-type virus and sub-20 nM activity against viral variants bearing Tyr181Cys and Lys103Asn mutations in HIV-RT. An X-ray crystal structure at 2.49 Å resolution is also reported for the key compound 6e with HIV-RT.

Research paper thumbnail of Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe

Nucleic Acids Research, 2003

Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sit... more Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution. Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors. In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3¢ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3¢-end of the 25 S rRNA sequence. In this study normal and mutant 3¢ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing. Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3¢ETS. The cleavage of mutant 3¢ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo. One mutant, however, was ef®ciently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro. In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3¢-end of the 25 rRNA sequence, indicating that the removal of the 3¢ETS does not require cleavage at the intermediate sites. These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identi®ed by analyses of termini, and/or processing, in vitro.

Research paper thumbnail of Pre-18S Ribosomal RNA Is Structurally Compacted into the SSU Processome Prior to Being Cleaved from Nascent Transcripts in Saccharomyces cerevisiae

Molecular Cell, 2004

Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosom... more Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.

Research paper thumbnail of Picomolar Inhibitors of HIV Reverse Transcriptase Featuring Bicyclic Replacement of a Cyanovinylphenyl Group

Journal of the American Chemical Society, 2013

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 revers... more Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC50 values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds such as rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4 nM activity against the wild-type virus. The compounds also performed well with EC50 values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogues were also investigated; the most potent compounds in these cases have EC50 values toward wild-type HIV-1 near 10 nM and high-nanomolar activities toward the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88 Å resolution for the complex of the parent 2-cyanoindolizine 10b and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogues were also measured to be ~40 μg/mL, which is similar to that for the approved drug efavirenz and ~1000-fold greater than for rilpivirine.

Research paper thumbnail of Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB

Journal of Structural Biology, 2010

FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we... more FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we report the crystal structures of the intracellular domain of FeoB (NFeoB) from Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus (PfNFeoB) with and without bound ligands. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (Sdomain), which despite its lack of sequence similarity between species is structurally conserved. In the nucleotide-free state, the G-domain's two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays important roles in FeoB function.

Research paper thumbnail of Characterization of a Novel Prokaryotic GDP Dissociation Inhibitor Domain from the G Protein Coupled Membrane Protein FeoB

Journal of Molecular Biology, 2008

The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in ... more The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in prokaryotes. Here, we report that FeoB harbors a novel GDP dissociation inhibitor like domain that specifically stabilizes GDP-binding through an interaction with the switch I region of the G protein. We show that the stabilization of GDP-binding is conserved between species despite a high degree of sequence variability in their GDI-like domains, and demonstrate that the presence of the membrane embedded domain increases GDP-binding affinity roughly 150-fold over the level accomplished by action of the GDI-like domain alone. To our knowledge, this is the first example for a prokaryotic GDI, targeting a bacterial G protein-coupled membrane process. Our findings suggest that Fe(II) uptake in bacteria involves a G protein regulatory pathway reminiscent of signaling mechanisms found in higher order organisms.

Research paper thumbnail of Structure-Based Evaluation of C5 Derivatives in the Catechol Diether Series Targeting HIV-1 Reverse Transcriptase

Chemical Biology & Drug Design, 2014

Using a computationally driven approach, a class of inhibitors with picomolar potency known as th... more Using a computationally driven approach, a class of inhibitors with picomolar potency known as the catechol diethers were developed targeting the non-nucleoside-binding pocket of HIV-1 reverse transcriptase. Computational studies suggested that halogen-bonding interactions between the C5 substituent of the inhibitor and backbone carbonyl of conserved residue Pro95 might be important. While the recently reported crystal structures of the reverse transcriptase complexes confirmed the interactions with the non-nucleoside-binding pocket, they revealed the lack of a halogen-bonding interaction with Pro95. To understand the effects of substituents at the C5 position, we determined additional crystal structures with 5-Br and 5-H derivatives. Using comparative structural analysis, we identified several conformations of the ethoxy uracil dependent on the strength of a van der Waals interaction with the Cγ of Pro95 and the C5 substitution. The 5-Cl and 5-F derivatives position the ethoxy uracil to make more hydrogen bonds, whereas the larger 5-Br and smaller 5-H position the ethoxy uracil to make fewer hydrogen bonds. EC50 values correlate with the trends observed in the crystal structures. The influence of C5 substitutions on the ethoxy uracil conformation may have strategic value, as future derivatives can possibly be modulated to gain additional hydrogen-bonding interactions with resistant variants of reverse transcriptase.

Research paper thumbnail of Structural Basis of Membrane Invagination by F-BAR Domains

Cell, 2008

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structure... more BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.

Research paper thumbnail of Eastern extension of azoles as non-nucleoside inhibitors of HIV-1 reverse transcriptase; cyano group alternatives

Bioorganic & Medicinal Chemistry Letters, 2010

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with the assi... more Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with the assistance of free energy perturbation (FEP) calculations to predict relative free energies of binding. Extension of azole-containing inhibitors into an 'eastern' channel between Phe227 and Pro236 has led to the discovery of potent and structurally novel derivatives.

Research paper thumbnail of RAC protein induces enzymatic access to the maturing 3′-end of the 25S rRNA in Schizosaccharomyces pombe

Biochemical and Biophysical Research Communications, 2008

In Schizosaccharomyces pombe, interdependency between steps in the processing of the rRNAs is med... more In Schizosaccharomyces pombe, interdependency between steps in the processing of the rRNAs is mediated by a large protein complex (RAC) which interacts with the non-conserved transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of Pac1 nuclease cleavage, leading to the removal of the 3' external transcribed spacers (3'ETS) in the maturation of the 3'ETS region. In this study modification exclusion and S1 nuclease were used to probe the RAC protein binding site and any subsequent structural changes in the maturing region. The results indicate that, as previously observed with the ITS1 and ITS2 regions, the upper helical region in the highly conserved extended terminal hairpin constitutes a protein binding site. In turn, this interaction induces a conformational change which affords access to nuclease at the 3'-end of the maturing 25S rRNA sequence.

Research paper thumbnail of Extension into the entrance channel of HIV-1 reverse transcriptase—Crystallography and enhanced solubility

Bioorganic & Medicinal Chemistry Letters, 2013

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature exten... more Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent.

Research paper thumbnail of DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function

Molecular and cellular biology, 2015

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs... more DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, i...

Research paper thumbnail of Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase

Bioorganic & Medicinal Chemistry Letters, 2015

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a... more Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

Research paper thumbnail of DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function

Molecular and cellular biology, 2015

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs... more DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, i...

Research paper thumbnail of Structure-Based Evaluation of Non-nucleoside Inhibitors with Improved Potency and Solubility That Target HIV Reverse Transcriptase Variants

Journal of medicinal chemistry, Jan 5, 2015

The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV... more The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 μg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of comp...

Research paper thumbnail of Illuminating HIV gp120-ligand recognition through computationally-driven optimization of antibody-recruiting molecules

Chemical Science, 2014

Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV... more Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV gp120 (ARM-H). These studies have leveraged a combination of medicinal chemistry, biochemical and cellular assay analysis, and computation. Our findings have afforded an optimized analog of ARM-H, which is ~1000 fold more potent in gp120-binding and MT-2 antiviral assays than our previously reported derivative. Furthermore, computational analysis, taken together with experimental data, provides evidence that azaindole- and indole-based attachment inhibitors bind gp120 at an accessory hydrophobic pocket beneath the CD4-binding site and can also adopt multiple unique binding modes in interacting with gp120. These results are likely to prove highly enabling in the development of novel HIV attachment inhibitors, and more broadly, they suggest novel applications for ARMs as probes of conformationally flexible systems.

Research paper thumbnail of RAC Protein Directs the Complete Removal of the 3′ External Transcribed Spacer by the Pac1 Nuclease

Molecular Cell, 2002

In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein c... more In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS. Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence. The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.

Research paper thumbnail of Picomolar Inhibitors of HIV-1 Reverse Transcriptase: Design and Crystallography of Naphthyl Phenyl Ethers

ACS Medicinal Chemistry Letters, 2014

Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nuc... more Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Promising compounds are reported that show midpicomolar activity against the wild-type virus and sub-20 nM activity against viral variants bearing Tyr181Cys and Lys103Asn mutations in HIV-RT. An X-ray crystal structure at 2.49 Å resolution is also reported for the key compound 6e with HIV-RT.

Research paper thumbnail of Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe

Nucleic Acids Research, 2003

Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sit... more Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution. Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors. In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3¢ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3¢-end of the 25 S rRNA sequence. In this study normal and mutant 3¢ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing. Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3¢ETS. The cleavage of mutant 3¢ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo. One mutant, however, was ef®ciently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro. In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3¢-end of the 25 rRNA sequence, indicating that the removal of the 3¢ETS does not require cleavage at the intermediate sites. These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identi®ed by analyses of termini, and/or processing, in vitro.

Research paper thumbnail of Pre-18S Ribosomal RNA Is Structurally Compacted into the SSU Processome Prior to Being Cleaved from Nascent Transcripts in Saccharomyces cerevisiae

Molecular Cell, 2004

Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosom... more Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.

Research paper thumbnail of Picomolar Inhibitors of HIV Reverse Transcriptase Featuring Bicyclic Replacement of a Cyanovinylphenyl Group

Journal of the American Chemical Society, 2013

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 revers... more Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC50 values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds such as rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4 nM activity against the wild-type virus. The compounds also performed well with EC50 values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogues were also investigated; the most potent compounds in these cases have EC50 values toward wild-type HIV-1 near 10 nM and high-nanomolar activities toward the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88 Å resolution for the complex of the parent 2-cyanoindolizine 10b and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogues were also measured to be ~40 μg/mL, which is similar to that for the approved drug efavirenz and ~1000-fold greater than for rilpivirine.

Research paper thumbnail of Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB

Journal of Structural Biology, 2010

FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we... more FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we report the crystal structures of the intracellular domain of FeoB (NFeoB) from Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus (PfNFeoB) with and without bound ligands. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (Sdomain), which despite its lack of sequence similarity between species is structurally conserved. In the nucleotide-free state, the G-domain's two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays important roles in FeoB function.

Research paper thumbnail of Characterization of a Novel Prokaryotic GDP Dissociation Inhibitor Domain from the G Protein Coupled Membrane Protein FeoB

Journal of Molecular Biology, 2008

The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in ... more The FeoB family of membrane embedded G proteins are involved with high affinity Fe(II) uptake in prokaryotes. Here, we report that FeoB harbors a novel GDP dissociation inhibitor like domain that specifically stabilizes GDP-binding through an interaction with the switch I region of the G protein. We show that the stabilization of GDP-binding is conserved between species despite a high degree of sequence variability in their GDI-like domains, and demonstrate that the presence of the membrane embedded domain increases GDP-binding affinity roughly 150-fold over the level accomplished by action of the GDI-like domain alone. To our knowledge, this is the first example for a prokaryotic GDI, targeting a bacterial G protein-coupled membrane process. Our findings suggest that Fe(II) uptake in bacteria involves a G protein regulatory pathway reminiscent of signaling mechanisms found in higher order organisms.

Research paper thumbnail of Structure-Based Evaluation of C5 Derivatives in the Catechol Diether Series Targeting HIV-1 Reverse Transcriptase

Chemical Biology & Drug Design, 2014

Using a computationally driven approach, a class of inhibitors with picomolar potency known as th... more Using a computationally driven approach, a class of inhibitors with picomolar potency known as the catechol diethers were developed targeting the non-nucleoside-binding pocket of HIV-1 reverse transcriptase. Computational studies suggested that halogen-bonding interactions between the C5 substituent of the inhibitor and backbone carbonyl of conserved residue Pro95 might be important. While the recently reported crystal structures of the reverse transcriptase complexes confirmed the interactions with the non-nucleoside-binding pocket, they revealed the lack of a halogen-bonding interaction with Pro95. To understand the effects of substituents at the C5 position, we determined additional crystal structures with 5-Br and 5-H derivatives. Using comparative structural analysis, we identified several conformations of the ethoxy uracil dependent on the strength of a van der Waals interaction with the Cγ of Pro95 and the C5 substitution. The 5-Cl and 5-F derivatives position the ethoxy uracil to make more hydrogen bonds, whereas the larger 5-Br and smaller 5-H position the ethoxy uracil to make fewer hydrogen bonds. EC50 values correlate with the trends observed in the crystal structures. The influence of C5 substitutions on the ethoxy uracil conformation may have strategic value, as future derivatives can possibly be modulated to gain additional hydrogen-bonding interactions with resistant variants of reverse transcriptase.

Research paper thumbnail of Structural Basis of Membrane Invagination by F-BAR Domains

Cell, 2008

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structure... more BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.

Research paper thumbnail of Eastern extension of azoles as non-nucleoside inhibitors of HIV-1 reverse transcriptase; cyano group alternatives

Bioorganic & Medicinal Chemistry Letters, 2010

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with the assi... more Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with the assistance of free energy perturbation (FEP) calculations to predict relative free energies of binding. Extension of azole-containing inhibitors into an 'eastern' channel between Phe227 and Pro236 has led to the discovery of potent and structurally novel derivatives.

Research paper thumbnail of RAC protein induces enzymatic access to the maturing 3′-end of the 25S rRNA in Schizosaccharomyces pombe

Biochemical and Biophysical Research Communications, 2008

In Schizosaccharomyces pombe, interdependency between steps in the processing of the rRNAs is med... more In Schizosaccharomyces pombe, interdependency between steps in the processing of the rRNAs is mediated by a large protein complex (RAC) which interacts with the non-conserved transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of Pac1 nuclease cleavage, leading to the removal of the 3' external transcribed spacers (3'ETS) in the maturation of the 3'ETS region. In this study modification exclusion and S1 nuclease were used to probe the RAC protein binding site and any subsequent structural changes in the maturing region. The results indicate that, as previously observed with the ITS1 and ITS2 regions, the upper helical region in the highly conserved extended terminal hairpin constitutes a protein binding site. In turn, this interaction induces a conformational change which affords access to nuclease at the 3'-end of the maturing 25S rRNA sequence.

Research paper thumbnail of Extension into the entrance channel of HIV-1 reverse transcriptase—Crystallography and enhanced solubility

Bioorganic & Medicinal Chemistry Letters, 2013

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature exten... more Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent.