scholarly journals Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

2016 ◽  
Vol 113 (33) ◽  
pp. 9274-9279 ◽  
Author(s):  
Zhe Li Salie ◽  
Karen A. Kirby ◽  
Eleftherios Michailidis ◽  
Bruno Marchand ◽  
Kamalendra Singh ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Complex Structure ◽  
Primer Binding Site ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Catalytic Complex ◽  
Hiv Reverse Transcriptase ◽  
A Chain ◽  
Ordered Water

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3′-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MPP•dT-MPN), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•EFdA-MPN); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MPP•EFdA-MP*N). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4′-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

Structural Basis for the Inhibition Mechanism of Ecotin against Neutrophil Elastase by Targeting the Active Site and Secondary Binding Site

Biochemistry ◽  
2020 ◽  
Vol 59 (30) ◽  
pp. 2788-2795
Author(s):  
Chacko Jobichen ◽  
Mahalakshmi Tirumuru Prabhakar ◽  
Su Ning Loh ◽  
J. Sivaraman
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Neutrophil Elastase ◽  
Structural Basis ◽  
Inhibition Mechanism

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

Selectivity of the surface binding site (SBS) on barley starch synthase I

Biologia ◽  
2014 ◽  
Vol 69 (9) ◽  
Author(s):  
Casper Wilkens ◽  
Jose Cuesta-Seijo ◽  
Monica Palcic ◽  
Birte Svensson
Keyword(s):  
Crystal Structure ◽  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Active Site ◽  
Mutational Analysis ◽  
Starch Synthase ◽  
Surface Binding ◽  
Binding Studies ◽  
A Chain ◽  
Surface Binding Site

AbstractStarch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable K D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.

scholarly journals Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
K. Thangavelu ◽  
Qing Yun Chong ◽  
Boon Chuan Low ◽  
J. Sivaraman
Keyword(s):  
Active Site ◽  
Human Kidney ◽  
Structural Basis ◽  
Inhibition Mechanism

Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody

2013 ◽  
Vol 450 (3) ◽  
pp. 477-486 ◽  
Author(s):  
Jean S. Sohier ◽  
Clémentine Laurent ◽  
Andy Chevigné ◽  
Els Pardon ◽  
Vasundara Srinivasan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Active Site ◽  
Substrate Inhibition ◽  
Inhibition Mechanism ◽  
Bacterial Strains ◽  
Single Domain Antibody ◽  
Alanine Scan ◽  
Hydrophobic Amino Acids ◽  
Micromolar Range

MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.

scholarly journals Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

2021 ◽  
Author(s):  
Kyle S. Skalenko ◽  
Lingting Li ◽  
Yuanchao Zhang ◽  
Irina O. Vvedenskaya ◽  
Jared T. Winkelman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Primer Binding Site ◽  
Structural Basis ◽  
Structural Description ◽  
Large Set ◽  
Promoter Sequences ◽  
E Coli

AbstractChemical modifications of RNA 5′ ends enable “epitranscriptomic” regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates (NTPs) for use as initiating entities, providing an ab initio mechanism for altering the RNA 5′ end. In Escherichia coli cells, RNAs with a 5′-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, “primer-dependent initiation.” Here we use massively systematic transcript end readout (“MASTER”) to detect and quantify RNA 5′ ends generated by primer-dependent initiation for ~410 (~1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through inter-chain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally-encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position, but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

scholarly journals Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weijie You ◽  
Wei Zheng ◽  
Sandra Weiss ◽  
Katrin F. Chua ◽  
Clemens Steegborn
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Specific Binding ◽  
Inhibitory Effect ◽  
Structural Basis ◽  
Allosteric Site ◽  
Quercetin Derivatives ◽  
Dependent Protein ◽  
Targeted Drug ◽  
Related Compounds

AbstractMammalian Sirtuin 6 (Sirt6) is an NAD+-dependent protein deacylase regulating metabolism and chromatin homeostasis. Sirt6 activation protects against metabolic and aging-related diseases, and Sirt6 inhibition is considered a cancer therapy. Available Sirt6 modulators show insufficient potency and specificity, and even partially contradictory Sirt6 effects were reported for the plant flavone quercetin. To understand Sirt6 modulation by quercetin-based compounds, we analysed their binding and activity effects on Sirt6 and other Sirtuin isoforms and solved crystal structures of compound complexes with Sirt6 and Sirt2. We find that quercetin activates Sirt6 via the isoform-specific binding site for pyrrolo[1,2-a]quinoxalines. Its inhibitory effect on other isoforms is based on an alternative binding site at the active site entrance. Based on these insights, we identified isoquercetin as a ligand that can discriminate both sites and thus activates Sirt6 with increased specificity. Furthermore, we find that quercetin derivatives that inhibit rather than activate Sirt6 exploit the same general Sirt6 binding site as the activators, identifying it as a versatile allosteric site for Sirt6 modulation. Our results thus provide a structural basis for Sirtuin effects of quercetin-related compounds and helpful insights for Sirt6-targeted drug development.

scholarly journals The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

2008 ◽  
Vol 82 (19) ◽  
pp. 9577-9590 ◽  
Author(s):  
Arnaud Gruez ◽  
Barbara Selisko ◽  
Michael Roberts ◽  
Gérard Bricogne ◽  
Cécile Bussetta ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Binding Site ◽  
Complex Structure ◽  
Coxsackievirus B3 ◽  
Primer Binding Site ◽  
Genome Replication ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Mouth Disease Virus

ABSTRACT The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3Dpol) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

scholarly journals Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 371 ◽  
Author(s):  
Xiao-Ping Li ◽  
Jennifer Kahn ◽  
Nilgun Tumer
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Active Site ◽  
Ribosome Binding Site ◽  
Ricin A Chain ◽  
Ribosome Binding ◽  
P Proteins ◽  
A Chain ◽  
Ribosomal P ◽  
Ricin A

Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) by interacting with the C-termini of the ribosomal P stalk. The ribosome interaction site and the active site are located on opposite faces of RTA. The interaction with P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high activity by orienting the active site towards the SRL. Therefore, if an inhibitor disrupts RTA–ribosome interaction by binding to the ribosome binding site of RTA, it should inhibit the depurination activity. To test this model, we synthesized peptides mimicking the last 3 to 11 amino acids of P proteins and examined their interaction with wild-type RTA and ribosome binding mutants by Biacore. We measured the inhibitory activity of these peptides on RTA-mediated depurination of yeast and rat liver ribosomes. We found that the peptides interacted with the ribosome binding site of RTA and inhibited depurination activity by disrupting RTA–ribosome interactions. The shortest peptide that could interact with RTA and inhibit its activity was four amino acids in length. RTA activity was inhibited by disrupting its interaction with the P stalk without targeting the active site, establishing the ribosome binding site as a new target for inhibitor discovery.

scholarly journals Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Renhong Yan ◽  
Yaning Li ◽  
Jennifer Müller ◽  
Yuanyuan Zhang ◽  
Simon Singer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Binding ◽  
Complex Structure ◽  
Binding Pocket ◽  
Structural Basis ◽  
Inhibition Mechanism ◽  
Amino Acid Transporters ◽  
Future Drug ◽  
First Time ◽  
The Brain

AbstractLAT1 (SLC7A5) is one of the representative light chain proteins of heteromeric amino acid transporters, forming a heterodimer with its heavy chain partner 4F2hc (SLC3A2). LAT1 is overexpressed in many types of tumors and mediates the transfer of drugs and hormones across the blood-brain barrier. Thus, LAT1 is considered as a drug target for cancer treatment and may be exploited for drug delivery into the brain. Here, we synthesized three potent inhibitors of human LAT1, which inhibit transport of leucine with IC50 values between 100 and 250 nM, and solved the cryo-EM structures of the corresponding LAT1-4F2hc complexes with these inhibitors bound at resolution of up to 2.7 or 2.8 Å. The protein assumes an outward-facing occluded conformation, with the inhibitors bound in the classical substrate binding pocket, but with their tails wedged between the substrate binding site and TM10 of LAT1. We also solved the complex structure of LAT1-4F2hc with 3,5-diiodo-l-tyrosine (Diiodo-Tyr) at 3.4 Å overall resolution, which revealed a different inhibition mechanism and might represent an intermediate conformation between the outward-facing occluded state mentioned above and the outward-open state. To our knowledge, this is the first time that the outward-facing conformation is revealed for the HAT family. Our results unveil more important insights into the working mechanisms of HATs and provide a structural basis for future drug design.

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