Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography. The UL53 structures, determined at 1.93 and 3.0 Å resolution, contained unexpected features including a Bergerat fold resembling that found in certain nucleotide-binding proteins, and a Cys 3 His zinc finger. Substitutions of zinc-coordinating residues decreased UL50-UL53 co-localization in transfected cells, and, when incorporated into the HCMV genome, ablated viral replication. The structure of the complex, determined at 2.47 Å resolution, revealed a mechanism of heterodimerization in which UL50 clamps onto helices of UL53 like a vise. Substitutions of particular residues on the interaction interface disrupted UL50-UL53 colocalization in transfected cells and abolished virus production. The structures and the identification of contacts can be harnessed toward the rational design of novel and highly specific antiviral drugs and will aid in the detailed understanding of nuclear egress.

Background: ZO-1 is a scaffolding protein implicated in the assembly of tight junctions. Results: Structures of core PDZ-SH3-GUK, plus and minus JAM-A peptide, and isolated PDZ are presented. Conclusion: The SH3 domain is required for JAM-A binding to PDZ3. Significance: This is the first demonstration for the role of an adjacent domain to the binding of ligands to PDZ domains in the MAGUK family.

Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses. Herpesviruses replicate and package their DNA genomes into capsids in the nucleus of the host cell. The nucleocapsids are then transported out of the nucleus through an unusual process called nuclear egress. A widely accepted model for nuclear egress entails envelopment and de-envelopment of capsids as they transit the nuclear membranes (reviewed in references 1, 2, and 3). Studies of human cytomegalovirus (HCMV) nuclear egress are of particular interest, because of the medical ...

ZO-1 is a multidomain protein involved in cell-cell junctions and contains three PDZ domains, which are necessary for its function in vivo. PDZ domains play a central role in assembling diverse protein complexes through their ability to recognize short peptide motifs on other proteins. We determined the structure of the second of the three PDZ domains of ZO-1, which is known to promote dimerization as well as bind to C-terminal sequences on connexins. The dimer is stabilized by extensive symmetrical domain swapping of ␤-strands, which is unlike any other known mechanism of PDZ dimerization. The canonical peptide-binding groove remains intact in both subunits of the PDZ2 dimer and is created by elements contributed from both monomers. This unique structure reveals an additional example of how PDZ domains dimerize and has multiple implications for both peptide binding and oligomerization in vivo.PDZ (PSD-95/Discs Large/zonula occludens-1) domains are modular protein-binding motifs that are found in bacteria, plants, and animals. They play a key role in scaffolding protein complexes through their ability to recognize short polypeptide motifs on other proteins and in some cases their ability to crosslink through dimerization. Although found in proteins with functions as diverse as cell signaling, cytoskeletal structure, cell polarity, and trafficking, many PDZ proteins are associated with the plasma membrane, where they mediate the assembly of specific subcellular domains like synapses and cell-cell junctions (reviewed in Ref. 1). Thus, the mechanisms for peptide binding and dimerization of PDZ domains have important implications for several fields.Although quite diverse at the primary sequence level, Ͼ150 known PDZ domain structures all share a conserved globular cluster of 5-6 ␤-strands and 1-2 ␣-helices (2). Peptide binding is mediated by a surface groove formed by an antiparallel ␤-strand (␤2) and ␣-helix (␣2) ((2) and reviewed in (3)). The majority of PDZ domain ligands are C-terminal peptides, which lie in this surface groove and form main chain interactions with ␣2 and ␤2. These interactions are highly specific and dependent on the terminal 3-4 amino acids of the peptide ligand (4). However, other residues within the ligand may also contribute to the specificity and affinity of the PDZ-ligand interaction (5). In addition, other binding modalities have also been characterized. For example, some PDZ domains can bind to internal peptide sequences or phosphoinositides (6 -8). Finally, a limited number of PDZ domains can directly bind another PDZ domain to form homo-and hetero-oligomers (9 -12). In contrast to the conserved mechanism of C-terminal peptide binding, there exist several distinct mechanisms for dimerization.PDZ domains are particularly important in the organization of cellular tight junctions (TJ).3 Tight junctions form the paracellular barrier to the movement of ions, macromolecules, and cells across both endothelia and epithelia (13). Although several different classes of PDZ proteins are localized ...

Tight junctions are dynamic components of epithelial and endothelial cells that regulate the paracellular transport of ions, solutes, and immune cells. The assembly and permeability of these junctions is dependent on the zonula occludens (ZO) proteins, members of the membrane-associated guanylate kinase homolog (MAGUK) protein family, which are characterized by a core Src homology 3 (SH3)-GUK module that coordinates multiple protein-protein interactions. The structure of the ZO-1 SH3-GUK domain confirms that the interdependent folding of the SH3 and GUK domains is a conserved feature of MAGUKs, but differences in the orientation of the GUK domains in three different MAGUKs reveal interdomain flexibility of the core unit. Using pull-down assays, we show that an effector loop, the U6 region in ZO-1, forms a novel intramolecular interaction with the core module. This interaction is divalent cation-dependent and overlaps with the binding site for the regulatory molecule calmodulin on the GUK domain. These findings provide insight into the previously observed ability of the U6 region to regulate TJ assembly in vivo and the structural basis for the complex protein interactions of the MAGUK family.The assembly and functional organization of plasma membrane domains, such as synapses and cell adhesions, depends on the temporally and spatially regulated assembly of multiprotein complexes. In one common paradigm, the trafficking and assembly of the transmembrane receptors and adhesion molecules that form these complexes is organized by a series of cytosolic scaffolding proteins. One of the most common and ubiquitous families of scaffolding molecules is the membraneassociated guanylate kinase homologs (MAGUKs).2 Founding members of this family include the discs large tumor suppressor in Drosophila, Lin2, which is required for RT kinase signaling in Caenorhabditis and PSD-95 and its related synaptic scaffolding proteins, which also control channel activity. This diverse family of proteins is characterized by a core motif of protein-binding domains, including a PSD-95/DLG/ZO-1 (PDZ) domain, an Src homology 3 (SH3) domain, and a region of homology to guanylate kinase referred to as the GUK domain ( Fig. 1A) (reviewed in Ref. 1). Most also include additional PDZ domains or other conserved protein-binding domains. These domains are separated by unique regions (U regions), which can also have protein-binding and regulatory roles (1, 2). However, it is the core SH3-GUK module and its adjacent unique domains that often have a dominant role in the regulation of transmembrane ligand binding, protein cross-linking, and localization to the appropriate subcellular domain.The structural basis for the regulatory properties of the various MAGUKs is poorly understood. To date, the only core motif that has been resolved at an atomic level belongs to PSD-95 (postsynaptic density protein 95) (3, 4), a neuronal protein involved in the regulation of postsynaptic gated ion channel. The structure reveals that the SH3 and GUK domains are not simply...