[ Autophagy 4:8, 989-997; 16 November 2008]; ©2008 Landes Bioscience γ-Herpesviruses (γHVs), including important human pathogens such as Epstein Barr virus, Kaposi's sarcoma-associated HV, and the murine γHV68, encode homologs of the antiapoptotic, cellular Bcl-2 (cBcl-2) to promote viral replication and pathogenesis. The precise molecular details by which these proteins function in viral infection are poorly understood. Autophagy, a lysosomal degradation pathway, is inhibited by the interaction of cBcl-2s with a key autophagy effector, Beclin 1, and can also be inhibited by γHV Bcl-2s. Here we investigate the γHV68 M11-Beclin 1 interaction in atomic detail, using biochemical and structural approaches. We show that the Beclin 1 BH3 domain is the primary determinant of binding to M11 and other Bcl-2s, and this domain binds in a hydrophobic groove on M11, reminiscent of the binding of different BH3 domains to other Bcl-2s. Unexpectedly, regions outside of, but contiguous with, the Beclin 1 BH3 domain also contribute to this interaction. We find that M11 binds to Beclin 1 more strongly than do KSHV Bcl-2 or cBcl-2. Further, the differential affinity of M11 for different BH3 domains is caused by subtle, yet significant, variations in the atomic details of each interaction. Consistent with our structural analysis, we find that Beclin 1 residues L116 and F123, and M11 residue pairs G86 + R87 and Y60 + L74, are required for M11 to bind to Beclin 1 and downregulate autophagy. Thus, our results suggest that M11 inhibits autophagy through a mechanism that involves the binding of the Beclin 1 BH3 domain in the M11 hydrophobic surface groove.

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Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include intrinsically disordered regions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein-protein interactions, and delineate the network of proteins that interact with each predicted IDR-containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α-helix upon binding to BCL2s, with the C-terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in-depth investigations of the biological role and mechanism of IDRs in autophagy proteins.

Autophagy, an essential eukaryotic homeostasis pathway, enables sequestration of unwanted, damaged or harmful cytoplasmic components in vesicles called autophagosomes, enabling subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by Class III phosphatidylinositol 3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the anti-parallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered, but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for homodimerization, enabling us to identify ATG14 interface residues. Lastly, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on coimmunoprecipitation of the partner, and demonstrate that these mutations abrogate starvation-induced up-regulation of autophagy, but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer, and identifies interface residues important for BECN1:ATG14 heterodimerization and for autophagy.

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe 2 S 2 center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 Å. Steady-state kinetics assays demonstrated that BPDO had an apparent k cat /K m for biphenyl of (1. The microbial catabolic activities responsible for the degradation of aromatic compounds constitute an essential link in the global carbon cycle. These activities are of considerable practical interest due to their potential to destroy toxic, persistent pollutants, a strategy known as bioremediation (1). In the case of highly chlorinated, structurally diverse xenobiotics such as PCBs, 1 the development of a practical bioremediation technology has been limited in part by the failure of existing microbial catabolic activities to effectively degrade these compounds (1, 2). This failure may arise because these activities have not yet evolved to degrade compounds that have only recently been introduced into the biosphere. An important aspect of the adaptation of catabolic activities for bioremediation is the study of the structure and function of key catabolic enzymes. Such studies provide insight into the molecular basis of an important biological process, thereby facilitating the modification of enzyme specificity and the design of novel metabolic pathways.2BPDO catalyzes the initial reaction in the aerobic degradation of biphenyl and some PCBs. BPDO is a typical aromatic ring-hydroxylating dioxygenase, utilizing O 2 and electrons originating from NADH to transform biphenyl to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene ( Fig. 1) (3, 4). This dihydroxylation prepares the ring for subsequent degradation by ring cleavage enzymes. The enzyme comprises an FAD-containing reductase (BphG), a Rieske-type ferredoxin (BphF), and a two-subunit oxygenase of ␣ 3 ␤ 3 constitution that contains a Rieske-type Fe 2 S 2 cluster and a mononuclear iron center. Accordingly, BPDO has been classified as a group IIB aromatic ring-hydroxylating dioxygenase together with benzene and toluene dioxygenases (5). Structural and spectroscopic studies of related dioxygenases indicate that the mononuclear iron center orchestrates substrate transformation (reviewed in Ref. 6). BphG, BphF, and the oxygenase Fe 2 S 2 cluster function to transfer electrons from NADH to this center.BPDO is a major determinant of the PCB-catabolizing capabilities of biphenyl-degrading strains, and the enzymes from different strains possess significantly different congener-transforming abilities. For example, BPDO LB400 from Burkholderia cepacia LB400 transforms a much broader range of congeners than BPDO KF707 from Pseudomonas ps...