Crystal structure of the catalytic domain of HIV-1 restriction factor APOBEC3G in complex with ssDNA

The human APOBEC3G protein is a cytidine deaminase that generates cytidine to deoxy-uridine mutations in single-stranded DNA (ssDNA), and capable of restricting replication of HIV-1 by generating mutations in viral genome. The mechanism by which APOBEC3G specifically deaminates 5\u27-CC motifs has remained elusive since structural studies have been hampered due to apparently weak ssDNA binding of the catalytic domain of APOBEC3G. We overcame the problem by generating a highly active variant with higher ssDNA affinity. Here, we present the crystal structure of this variant complexed with a ssDNA substrate at 1.86 A resolution. This structure reveals atomic-level interactions by which APOBEC3G recognizes a functionally-relevant 5\u27-TCCCA sequence. This complex also reveals a key role of W211 in substrate recognition, implicating a similar recognition in activation-induced cytidine deaminase (AID) with a conserved tryptophan

Time series analysis of categorical data using auto-odds ratio function

10.1080/02331888.2017.1421196Statistics522426-44

Switching DNA-binding specificity by unnatural amino acid substitution

The specificity of protein–nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid–protein complex. We have used cI repressor of bacteriophage l as a model system. In the l-repressor–DNA complex, the «-NH2 group (hydrogen bond donor) of lysine-4 of l-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site OL1. Substitution of lysine-4 (two donors) by isosteric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of l-repressor from C:G to T:A at position 6 of OL1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificit

CD spectra of wild-type (solid line; 2 µM) and -(2-hydroxyethyl)-cysteine-4 λ-repressor (dashed line; 2

<p><b>Copyright information:</b></p><p>Taken from "Switching DNA-binding specificity by unnatural amino acid substitution"</p><p>Nucleic Acids Research 2005;33(18):5896-5903.</p><p>Published online 13 Oct 2005</p><p>PMCID:PMC1258173.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>4 µM). Experimental details are in Materials and Methods

Fluorescence emission spectra of wild-type (solid line; 2 µM) and -(2-hydroxyethyl)-cysteine-4 λ-repressor (dashed line; 2

<p><b>Copyright information:</b></p><p>Taken from "Switching DNA-binding specificity by unnatural amino acid substitution"</p><p>Nucleic Acids Research 2005;33(18):5896-5903.</p><p>Published online 13 Oct 2005</p><p>PMCID:PMC1258173.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>4 µM). Experimental details are in Materials and Methods

Crystal structure of λ-repressor N-terminal domain crystal structure complexed with O1

<p><b>Copyright information:</b></p><p>Taken from "Switching DNA-binding specificity by unnatural amino acid substitution"</p><p>Nucleic Acids Research 2005;33(18):5896-5903.</p><p>Published online 13 Oct 2005</p><p>PMCID:PMC1258173.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Lysine-4 is shown in green and asparagine-55 is shown in cyan. The base pair CG6 is shown in space filling representation. Lower panel depicts the sequence of O1. The top half is the consensus half. Of the two strands, the top of left strand is the 5′ end

Nickel-catalyzed hydrogenolysis of unactivated carbon–cyano bonds

Selective hydrogenolysis of C–CN bonds can allow chemists to take advantage of ortho-directing ability, α-C–H acidity and electron withdrawing ability of the cyano group for synthetic manipulations. We have discovered hydrogenolysis of aryl and aliphatic cyanides under just 1 bar of hydrogen by using a nickel catalyst. This protocol was applied in the aryl cyanide directed functionalization reaction and α-substitution of benzyl cyanides

Interactions of APOBEC3s with DNA and RNA

APOBEC3 enzymes are key enzymes in our innate immune system regulating antiviral response in HIV and unfortunately adding diversity in cancer as they deaminate cytosine. Seven unique single and double domain APOBEC3s provide them with unique activity and specificity profiles for this deamination. Recent crystal and NMR structures of APOBEC3 complexes are unraveling the variety of epitopes involved in binding nucleic acids, including at the catalytic site, elsewhere on the catalytic domain and in the inactive N-terminal domain. The interplay between these diverse interactions is critical to uncovering the mechanisms by which APOBEC3s recognize and process their substrates