Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Increasing the Stability of Flavin-Dependent Halogenases by Disulfide Engineering.

Flavin-dependent halogenases allow halogenation of electron-rich aromatic compounds under mild reaction conditions even at electronically unfavored positions with high regioselectivity. In order to expand the application of halogenases, the enzymes need to be improved in terms of stability and efficiency. A previous study with the tryptophan 6-halogenase Thal demonstrated that thermostable Thal variants tend to form dimers in solution while the wild type is present as a monomer. Based on this a dimeric Thal variant was generated that is covalently linked by disulfide bonds. Introducing two cysteine residues at the dimer interface resulted in the variant Thal CC with significantly increased thermostability (▵T50 =15.7 K) and stability over time at elevated temperature compared to the wild type. By introducing the homologous mutations into the tryptophan 5-halogenase PyrH, we were able to show that the stabilization by covalent dimerization can also be transferred to other halogenases. Moreover, it was possible to further increase the thermostability of PyrH by inserting cysteine mutations at alternative sites of the dimer interface.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF.

The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Šapart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.

The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctX.

Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX-SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.

Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes.

Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit.

Enzymatic Late-Stage Halogenation of Peptides.

The late-stage site-selective derivatisation of peptides has many potential applications in structure-activity relationship studies and postsynthetic modification or conjugation of bioactive compounds. The development of orthogonal methods for C-H functionalisation is crucial for such peptide derivatisation. Among them, biocatalytic methods are increasingly attracting attention. Tryptophan halogenases emerged as valuable catalysts to functionalise tryptophan (Trp), while direct enzyme-catalysed halogenation of synthetic peptides is yet unprecedented. Here, it is reported that the Trp 6-halogenase Thal accepts a wide range of amides and peptides containing a Trp moiety. Increasing the sequence length and reaction optimisation made bromination of pentapeptides feasible with good turnovers and a broad sequence scope, while regioselectivity turned out to be sequence dependent. Comparison of X-ray single crystal structures of Thal in complex with d-Trp and a dipeptide revealed a significantly altered binding mode for the peptide. The viability of this bioorthogonal approach was exemplified by halogenation of a cyclic RGD peptide.

Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate.

Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscVC) and Aeromonas hydrophila (AscVC) are reported. Self-rotation functions revealed that LscVC forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscVC was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers.

Direct interaction of a chaperone-bound type III secretion substrate with the export gate.

Several gram-negative bacteria employ type III secretion systems (T3SS) to inject effector proteins into eukaryotic host cells directly from the bacterial cytoplasm. The export gate SctV (YscV in Yersinia) binds substrate:chaperone complexes such as YscX:YscY, which are essential for formation of a functional T3SS. Here, we present structures of the YscX:YscY complex alone and bound to nonameric YscV. YscX binds its chaperone YscY at two distinct sites, resembling the heterotrimeric complex of the T3SS needle subunit with its chaperone and co-chaperone. In the ternary complex the YscX N-terminus, which mediates YscX secretion, occupies a binding site within one YscV that is also used by flagellar chaperones, suggesting the interaction's importance for substrate recognition. The YscX C-terminus inserts between protomers of the YscV ring where the stalk protein binds to couple YscV to the T3SS ATPase. This primary YscV-YscX interaction is essential for the formation of a secretion-competent T3SS.

A recurring packing contact in crystals of InlB pinpoints functional binding sites in the internalin domain and the B repeat.

InlB, a bacterial agonist of the human receptor tyrosine kinase MET, consists of an N-terminal internalin domain, a central B repeat and three C-terminal GW domains. In all previous structures of full-length InlB or an InlB construct lacking the GW domains (InlB392), there was no interpretable electron density for the B repeat. Here, three InlB392 crystal structures in which the B repeat is resolved are described. These are the first structures to reveal the relative orientation of the internalin domain and the B repeat. A wild-type structure and two structures of the T332E variant together contain five crystallographically independent molecules. Surprisingly, the threonine-to-glutamate substitution in the B repeat substantially improved the crystallization propensity and crystal quality of the T332E variant. The internalin domain and B repeat are quite rigid internally, but are flexibly linked to each other. The new structures show that inter-domain flexibility is the most likely cause of the missing electron density for the B repeat in previous InlB structures. A potential binding groove between B-repeat strand ß2 and an adjacent loop forms an important crystal contact in all five crystallographically independent chains. This region may represent a hydrophobic `sticky patch' that supports protein-protein interactions. This assumption agrees with the previous finding that all known inactivating point mutations in the B repeat lie within strand ß2. The groove formed by strand ß2 and the adjacent loop may thus represent a functionally important protein-protein interaction site in the B repeat.

Conformational changes of loops highlight a potential binding site in Rhodococcus equi VapB.

Virulence-associated proteins (Vaps) contribute to the virulence of the pathogen Rhodococcus equi, but their mode of action has remained elusive. All Vaps share a conserved core of about 105 amino acids that folds into a compact eight-stranded antiparallel ß-barrel with a unique topology. At the top of the barrel, four loops connect the eight ß-strands. Previous Vap structures did not show concave surfaces that might serve as a ligand-binding site. Here, the structure of VapB in a new crystal form was determined at 1.71 Šresolution. The asymmetric unit contains two molecules. In one of them, the loop regions at the top of the barrel adopt a different conformation from other Vap structures. An outward movement of the loops results in the formation of a hydrophobic cavity that might act as a ligand-binding site. This lends further support to the hypothesis that the structural similarity between Vaps and avidins suggests a potential binding function for Vaps.

Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis.

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

Structure of apo flavin-dependent halogenase Xcc4156 hints at a reason for cofactor-soaking difficulties.

Flavin-dependent halogenases regioselectively introduce halide substituents into electron-rich substrates under mild reaction conditions. For the enzyme Xcc4156 from Xanthomonas campestris, the structure of a complex with the cofactor flavin adenine dinucleotide (FAD) and a bromide ion would be of particular interest as this enzyme exclusively brominates model substrates in vitro. Apo Xcc4156 crystals diffracted to 1.6 Šresolution. The structure revealed an open substrate-binding site lacking the loop regions that close off the active site and contribute to substrate binding in tryptophan halogenases. Therefore, Xcc4156 might accept larger substrates, possibly even peptides. Soaking of apo Xcc4156 crystals with FAD led to crumbling of the intergrown crystals. Around half of the crystals soaked with FAD did not diffract, while in the others there was no electron density for FAD. The FAD-binding loop, which changes its conformation between the apo and the FAD-bound form in related enzymes, is involved in a crystal contact in the apo Xcc4156 crystals. The conformational change that is predicted to occur upon FAD binding would disrupt this crystal contact, providing a likely explanation for the destruction of the apo crystals in the presence of FAD. Soaking with only bromide did not result in bromide bound to the catalytic halide-binding site. Simultaneous soaking with FAD and bromide damaged the crystals more severely than soaking with only FAD. Together, these latter two observations suggest that FAD and bromide bind to Xcc4156 with positive cooperativity. Thus, apo Xcc4156 crystals provide functional insight into FAD and bromide binding, even though neither the cofactor nor the halide is visible in the structure.

Binding of FAD and tryptophan to the tryptophan 6-halogenase Thal is negatively coupled.

Flavin-dependent halogenases require reduced flavin adenine dinucleotide (FADH2 ), O2 , and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6-halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH2 by flavin reductases.

Competitive Binding Study Revealing the Influence of Fluorophore Labels on Biomolecular Interactions.

Fluorescence methods are important tools in modern biology. Direct labeling of biomolecules with a fluorophore might, however, change interaction surfaces. Here, we introduce a competitive binding assay in combination with fluorescence correlation spectroscopy that reports binding affinities of both labeled and unlabeled biomolecules to their binding target. We investigated how fluorophore labels at different positions of a DNA oligonucleotide affect hybridization to a complementary oligonucleotide and found dissociation constants varying within 2 orders of magnitude. We next demonstrated that placing a fluorophore label at position Leu280 in the protein ligand internalin B does not alter the binding affinity to the MET receptor tyrosine kinase, compared to unlabeled internalin B. Our approach is simple to implement and can be applied to investigate the influence of fluorophore labels in a large variety of biomolecular interactions.

Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering.

Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.

Specific high affinity interaction of Helicobacter pylori CagL with integrin αV ß6 promotes type IV secretion of CagA into human cells.

CagL is an essential pilus surface component of the virulence-associated type IV secretion system (T4SS) employed by Helicobacter pylori to translocate the oncogenic effector protein CagA into human gastric epithelial cells. CagL contains an RGD motif and integrin α5 ß1 is widely accepted as its host cell receptor. Here, we show that CagL binds integrin αV ß6 with substantially higher affinity and that this interaction is functionally important. Cell surface expression of αV ß6 on various cell lines correlated perfectly with cell adhesion to immobilized CagL and with binding of soluble CagL to cells. We found no such correlation for α5 ß1 . The purified αV ß6 ectodomain bound CagL with high affinity. This interaction was highly specific, as the affinity of CagL for other RGD-binding integrins was two to three orders of magnitude weaker. Mutation of either conserved leucine in the CagL RGDLXXL motif, a motif that generally confers specificity for integrin αV ß6 and αV ß8 , lowered the affinity of CagL for αV ß6 . Stable expression of αV ß6 in αV ß6 -negative but α5 ß1 -expressing human cells promoted two hallmarks of the functional H. pylori T4SS, namely translocation of CagA into host cells and induction of interleukin-8 secretion by host cells. These findings suggest that integrin αV ß6 , although not essential for T4SS function, represents an important host cell receptor for CagL.

Inhibition of the MET Kinase Activity and Cell Growth in MET-Addicted Cancer Cells by Bi-Paratopic Linking.

MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.

Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal.

Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH2, oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate trypto-phan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Šresolution. The structure revealed that the C6 of tryptophan is positioned next to the ϵ-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of l-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.

Perfect merohedral twinning combined with noncrystallographic symmetry potentially causes the failure of molecular replacement with low-homology search models for the flavin-dependent halogenase HalX from Xanthomonas campestris.

Flavin-dependent halogenases can be used as biocatalysts because they regioselectively halogenate their substrates under mild reaction conditions. New halogenases with novel substrate specificities will add to the toolbox of enzymes available to organic chemists. HalX, the product of the xcc-b100_4193 gene, is a putative flavin-dependent halogenase from Xanthomonas campestris. The enzyme was recombinantly expressed and crystallized in order to aid in identifying its hitherto unknown substrate. Native data collected to a resolution of 2.5 Šshowed indications of merohedral twinning in a hexagonal lattice. Attempts to solve the phase problem by molecular replacement failed. Here, a detailed analysis of the suspected twinning is presented. It is most likely that the crystals are trigonal (point group 3) and exhibit perfect hemihedral twinning so that they appear to be hexagonal (point group 6). As there are several molecules in the asymmetric unit, noncrystallographic symmetry may complicate twinning analysis and structure determination.