During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn 2+ ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) β-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture—the non-local cross-β structure connecting the two β-sheets—and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing a variety of severe viral infections, including irreversible congenital disabilities. Nowadays, HCMV infection is treated by inhibiting the viral DNA polymerase. However, DNA polymerase inhibitors have several drawbacks. An alternative strategy is to use compounds against the packaging machinery or terminase complex, which is essential for viral replication. Our discovery that raltegravir ( 1 ), a human immunodeficiency virus drug, inhibits the nuclease function of UL89, one of the protein subunits of the complex, prompted us to further develop terminase inhibitors. On the basis of the structure of 1 , a library of diketoacid (α,γ-DKA and β,δ-DKA) derivatives were synthesized and tested for UL89-C nuclease activity. The mode of action of α,γ-DKA derivatives on the UL89 active site was elucidated by using X-ray crystallography, molecular docking, and in vitro experiments. Our studies identified α,γ-DKA derivative 14 able to inhibit UL89 in vitro in the low micromolar range, making 14 an optimal candidate for further development and virus-infected cell assay.