The tetrameric assembly of 2-aminomuconic acid dehydrogenase is a functional requirement of cofactor NAD+ binding

The bacterium Pseudomonas sp. AP-3 is able to use the environmental pollutant 2-aminophenol as its sole source of carbon, nitrogen, and energy. Eight genes (amnA, B, C, D, E, F, G, and H) encoding 2-aminophenol metabolizing enzymes are clustered into a single operon. 2-aminomuconic 6-semialdehyde dehydrogenase (AmnC), a member of the aldehyde dehydrogenase (ALDH) superfamily, is responsible for oxidizing 2-aminomuconic 6-semialdehyde to 2-aminomuconate. In contrast to many other members of the ALDH superfamily, the structural basis of the catalytic activity of AmnC remains elusive. Here, we present the crystal structure of AmnC, which displays a homotetrameric quaternary assembly that is directly involved in its enzymatic activity. The tetrameric state of AmnC in solution was also presented using small-angle X-ray scattering. The tetramerization of AmnC is mediated by the assembly of a protruding hydrophobic beta-strand motif and residues V121 and S123 located in the NAD+-binding domain of each subunit. Dimeric mutants of AmnC dramatically lose NAD+ binding affinity and enzyme activity, indicating that tetrameric assembly of AmnC is required for oxidizing the unstable metabolic intermediate 2-aminomuconic 6-semialdehyde to 2-aminomuconic acid in the 2-aminophenol metabolism pathway.

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

ABSTRACTLight sensing allows organisms to adapt to constantly changing environmental factors. Phytochromes constitute a widespread biological photoreceptor family that typically interconvert between two photostates called Pr (red light-absorbing) and Pfr (far-red light-absorbing). Despite the vast structural information reported on phytochromes, the lack of full-length structures at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here we present three crystallographic structures from the plant pathogenXanthomonas campestrisvirulence regulator bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structural findings, combined with mutational, biochemical and computational studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level, from the isomerization of the chromophore and the β-sheet/α-helix tongue transition to the remodeling of the quaternary assembly of the protein.TEASERCrystal structures of the full-length bacteriophytochromeXccBphP in both Pr and Pfr states unveil photoswitching mechanism

Cardiolipin, conformation, and respiratory complex-dependent oligomerization of the major mitochondrial ADP/ATP carrier in yeast

The phospholipid cardiolipin has pleiotropic structural and functional roles that are collectively essential for mitochondrial biology. Yet, the molecular details of how this lipid supports the structure and function of proteins and protein complexes are poorly understood. To address this property of cardiolipin, we use the mitochondrial adenosine 5′-diphosphate/adenosine 5′-triphosphate carrier (Aac) as a model. Here, we have determined that cardiolipin is critical for both the tertiary and quaternary assembly of the major yeast Aac isoform Aac2 as well as its conformation. Notably, these cardiolipin-provided structural roles are separable. In addition, we show that multiple copies of Aac2 engage in shared complexes that are largely dependent on the presence of assembled respiratory complexes III and IV or respiratory supercomplexes. Intriguingly, the assembly state of Aac2 is sensitive to its transport-related conformation. Together, these results expand our understanding of the numerous structural roles provided by cardiolipin for mitochondrial membrane proteins.

Targeting the Protein Tunnels of the Urease Accessory Complex: A Theoretical Investigation

Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori. Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the HpUreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the HpUreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein–protein interfaces on the HpUreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein–protein interfaces on the HpUreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic β-oxidation trifunctional enzyme complexes

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

Binding properties of the quaternary assembly protein SPAG1

In cells, many constituents are able to assemble resulting in large macromolecular machineries possessing very specific biological and physiological functions, e.g. ribosome, spliceosome and proteasome. Assembly of such entities is commonly mediated by transient protein factors. SPAG1 is a multidomain protein, known to participate in the assembly of both the inner and outer dynein arms. These arms are required for the function of sensitive and motile cells. Together with RUVBL1, RUVBL2 and PIH1D2, SPAG1 is a key element of R2SP, a protein complex assisting the quaternary assembly of specific protein clients in a tissue-specific manner and associating with heat shock proteins (HSPs) and regulators. In this study, we have investigated the role of TPR domains of SPAG1 in the recruitment of HSP chaperones by combining biochemical assays, ITC, NMR spectroscopy and molecular dynamics (MD) simulations. First, we propose that only two, out of the three TPR domains, are able to recruit the protein chaperones HSP70 and HSP90. We then focused on one of these TPR domains and elucidated its 3D structure using NMR spectroscopy. Relying on an NMR-driven docking approach and MD simulations, we deciphered its binding interface with the C-terminal tails of both HSP70 and HSP90. Finally, we addressed the biological function of SPAG1 and specifically demonstrated that a SPAG1 sub-fragment, containing a putative P-loop motif, cannot efficiently bind and hydrolyze GTP in vitro. Our data challenge the interpretation of SPAG1 possessing GTPase activity. We propose instead that SPAG1 regulates nucleotide hydrolysis activity of the HSP and RUVBL1/2 partners.

Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R

In human cells, thymidylate synthase (TS) provides the only source of 2’-deoxythymidyne-5’-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.

Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK

AmtR belongs to the TetR family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen-starvation conditions inCorynebacterium glutamicum. AmtR regulates the expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acidsetc. The recognition of operator DNA by homodimeric AmtR is not regulated by small-molecule effectors as in other TetR-family members but by a trimeric adenylylated PII-type signal transduction protein named GlnK. The crystal structure of ligand-free AmtR (AmtRorth) has been solved at a resolution of 2.1 Å in space groupP21212. Comparison of its quaternary assembly with the previously solved native AmtR structure (PDB entry 5dy1) in a trigonal crystal system (AmtRtri) not only shows how a solvent-content reduction triggers a space-group switch but also suggests a model for how dimeric AmtR might stoichiometrically interact with trimeric adenylylated GlnK.

QuaBingo: A Prediction System for Protein Quaternary Structure Attributes Using Block Composition

Background. Quaternary structures of proteins are closely relevant to gene regulation, signal transduction, and many other biological functions of proteins. In the current study, a new method based on protein-conserved motif composition in block format for feature extraction is proposed, which is termed block composition.Results. The protein quaternary assembly states prediction system which combines blocks with functional domain composition, called QuaBingo, is constructed by three layers of classifiers that can categorize quaternary structural attributes of monomer, homooligomer, and heterooligomer. The building of the first layer classifier uses support vector machines (SVM) based on blocks and functional domains of proteins, and the second layer SVM was utilized to process the outputs of the first layer. Finally, the result is determined by the Random Forest of the third layer. We compared the effectiveness of the combination of block composition, functional domain composition, and pseudoamino acid composition of the model. In the 11 kinds of functional protein families, QuaBingo is 23% of Matthews Correlation Coefficient (MCC) higher than the existing prediction system. The results also revealed the biological characterization of the top five block compositions.Conclusions. QuaBingo provides better predictive ability for predicting the quaternary structural attributes of proteins.