Renos Savva

Nucleotide mimicry in the crystal structure of the uracil-DNA glycosylase–uracil glycosylase inhibitor protein complex

The Bacillus subtilis bacteriophages PBS-1 and PBS-2 protect their uracil-containing DNA by expressing an inhibitor protein (UGI) which inactivates the host uracil-DNA glycosylase (UDGase) base-excision repair enzyme. Also, PBS1/2 UGI efficiently inactivates UDGases from other biological sources, including the enzyme from herpes simplex virus type-1 (HSV-1). The crystal structure of the HSV-1 UDGase–PBS1 UGI complex at 2.7 Å reveals an α-β-α sandwich structure for UGI which interacts with conserved regions of UDGase involved in DNA binding, and directly mimics protein–DNA interactions observed in the UDGase–oligonucleotide complex. The inhibitor completely blocks access to the active site of UDGase, but makes no direct contact with the uracil-binding pocket itself.

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi’s inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

A structural and functional dissection of the cardiac stress response factor MS1.

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F‐actin binding ability, located to the C‐terminal half of the protein, which promotes stabilization of F‐actin in the cell thus releasing myocardin‐related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor. Initial attempts to purify the protein only resulted in heavily degraded samples that showed distinct bands on SDS gels, suggesting the presence of stable domains. Using a combination of combinatorial domain hunting and sequence analysis, a set of potential domains was identified. The C‐terminal half of the protein actually contains two independent F‐actin binding domains. The most C‐terminal fragment (294–375), named actin binding domain 2 (ABD2), is independently folded while a proximal fragment called ABD1 (193–296) binds to F‐actin with higher affinity than ABD2 (KD 2.21 ± 0.47 μM vs. 10.61 ± 0.7 μM), but is not structured by itself in solution. NMR interaction experiments show that it binds and folds in a cooperative manner to F‐actin, justifying the label of domain. The architecture of the MS1 C‐terminus suggests that ABD1 alone could completely fulfill the F‐actin binding function opening up the intriguing possibility that ABD2, despite its high level of conservation, could have developed other functions. Proteins 2012.

The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues

Abstract 2′,4′-Dideoxy-4′-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4′-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4′-oxygen in recognition and cleavage of dU residues in DNA.

A structurally conserved motif in γ-herpesvirus uracil-DNA glycosylases elicits duplex nucleotide-flipping

Abstract Efficient γ-herpesvirus lytic phase replication requires a virally encoded UNG-type uracil-DNA glycosylase as a structural element of the viral replisome. Uniquely, γ-herpesvirus UNGs carry a seven or eight residue insertion of variable sequence in the otherwise highly conserved minor-groove DNA binding loop. In Epstein–Barr Virus [HHV-4] UNG, this motif forms a disc-shaped loop structure of unclear significance. To ascertain the biological role of the loop insertion, we determined the crystal structure of Kaposi’s sarcoma-associated herpesvirus [HHV-8] UNG (kUNG) in its product complex with a uracil-containing dsDNA, as well as two structures of kUNG in its apo state. We find the disc-like conformation is conserved, but only when the kUNG DNA-binding cleft is occupied. Surprisingly, kUNG uses this structure to flip the orphaned partner base of the substrate deoxyuridine out of the DNA duplex while retaining canonical UNG deoxyuridine-flipping and catalysis. The orphan base is stably posed in the DNA major groove which, due to DNA backbone manipulation by kUNG, is more open than in other UNG–dsDNA structures. Mutagenesis suggests a model in which the kUNG loop is pinned outside the DNA-binding cleft until DNA docking promotes rigid structuring of the loop and duplex nucleotide flipping, a novel observation for UNGs.

A structural and functional dissection of the cardiac stress response factor MS1

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F‐actin binding ability, located to the C‐terminal half of the protein, which promotes stabilization of F‐actin in the cell thus releasing myocardin‐related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor. Initial attempts to purify the protein only resulted in heavily degraded samples that showed distinct bands on SDS gels, suggesting the presence of stable domains. Using a combination of combinatorial domain hunting and sequence analysis, a set of potential domains was identified. The C‐terminal half of the protein actually contains two independent F‐actin binding domains. The most C‐terminal fragment (294–375), named actin binding domain 2 (ABD2), is independently folded while a proximal fragment called ABD1 (193–296) binds to F‐actin with higher affinity than ABD2 (KD 2.21 ± 0.47 μM vs. 10.61 ± 0.7 μM), but is not structured by itself in solution. NMR interaction experiments show that it binds and folds in a cooperative manner to F‐actin, justifying the label of domain. The architecture of the MS1 C‐terminus suggests that ABD1 alone could completely fulfill the F‐actin binding function opening up the intriguing possibility that ABD2, despite its high level of conservation, could have developed other functions. Proteins 2012.