Structural studies of a serum amyloid A octamer that is primed to scaffold lipid nanodiscs.

Serum amyloid A (SAA) is a highly conserved acute-phase protein that plays roles in activating multiple pro-inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high-density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein-protein interaction sites on SAA. While high-resolution structures for lipid-free, or apo-, forms of SAA have been reported, their relationship with the HDL-bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC-MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid-free and lipid-bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring-like structures reported for lipid-free ApoA-I. These SAA octamers represent a previously uncharacterized structure for lipid-free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA-I. The SAA-lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid-free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid-containing particles such as HDL. This study suggests a new model for SAA-lipid interactions and provides new insight into how SAA might stabilize protein-lipid nanodiscs or even replace ApoA-I as a scaffold for HDL particles during inflammation.

A secreted bacterial protein protects bacteria from cationic antimicrobial peptides by entrapment in phase-separated droplets.

Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat Neisseria gonorrhoeae, secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, Moraxella bovis, that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like M. bovis, leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.

The RavA-ViaA chaperone complex modulates bacterial persistence through its association with the fumarate reductase enzyme.

Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.

Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease.

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.

The evolutionary background and functional consequences of the rs2071307 polymorphism in human tropoelastin.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient ß-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for ß-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

Sequence variants of human tropoelastin affecting assembly, structural characteristics and functional properties of polymeric elastin in health and disease.

Elastin is the polymeric protein responsible for the physiologically important properties of extensibility and elastic recoil of cardiovascular, pulmonary and many other tissues. In spite of significant advances in the understanding how monomeric tropoelastin is assembled into the polymeric elastic matrix, details of this assembly process are still lacking. In particular it is not clear how the various architectures and more subtle elastic properties required by diverse elastic tissues can arise from the protein product of a single gene. While monomeric tropoelastin has the intrinsic ability to self-assemble into fibrillar structures, it is clear that in vivo assembly is guided by interactions with cells and other matrix-associated components. In addition, the multiplicity of reported mRNA isoforms of human tropoelastin, if translated into protein variants, could modulate not only interactions with these matrix-associated components but also self-assembly and functional properties. Critical information identifying such protein isoforms of human tropoelastin is only now emerging from mass spectrometric studies. Increased levels of complexity of the assembly process provide additional opportunities for production of polymeric elastins with aberrant architectures and sub-optimal functional properties that could affect the longer-term structural integrity of elastic matrices. Biophysical techniques, such as SAXS, NMR and molecular dynamics, have provided a means to discern details of the effects of sequence variants, including both alternate splicing isoforms and genetic polymorphisms, on the dynamic flexibility of elastin required for its elastomeric properties. Such approaches promise to provide important new insights into the relationship between sequence, structural characteristics, assembly and functional properties of elastin in both health and disease.

FRET Analysis of the Promiscuous yet Specific Interactions of the HIV-1 Vpu Transmembrane Domain.

The Vpu protein of HIV-1 functions to downregulate cell surface localization of host proteins involved in the innate immune response to viral infection. For several target proteins, including the NTB-A and PVR receptors and the host restriction factor tetherin, this antagonism is carried out via direct interactions between the transmembrane domains (TMDs) of Vpu and the target. The Vpu TMD also modulates homooligomerization of this protein, and the tetherin TMD forms homodimers. The mechanism through which a single transmembrane helix is able to recognize and interact with a wide range of select targets that do not share known interaction motifs is poorly understood. Here we use Förster resonance energy transfer to characterize the energetics of homo- and heterooligomer interactions between the Vpu TMD and several target proteins. Our data show that target TMDs compete for interaction with Vpu, and that formation of each heterooligomer has a similar dissociation constant (Kd) and free energy of association to the Vpu homooligomer. This leads to a model in which Vpu monomers, Vpu homooligomers, and Vpu-target heterooligomers coexist, and suggests that the conserved binding surface of Vpu TMD has been selected for weak binding to multiple targets.

Direct observation of structure and dynamics during phase separation of an elastomeric protein.

Despite its growing importance in biology and in biomaterials development, liquid-liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical cross-linking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient ß-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.

Single nucleotide polymorphisms and domain/splice variants modulate assembly and elastomeric properties of human elastin. Implications for tissue specificity and durability of elastic tissue.

Polymeric elastin provides the physiologically essential properties of extensibility and elastic recoil to large arteries, heart valves, lungs, skin and other tissues. Although the detailed relationship between sequence, structure and mechanical properties of elastin remains a matter of investigation, data from both the full-length monomer, tropoelastin, and smaller elastin-like polypeptides have demonstrated that variations in protein sequence can affect both polymeric assembly and tensile mechanical properties. Here we model known splice variants of human tropoelastin (hTE), assessing effects on shape, polymeric assembly and mechanical properties. Additionally we investigate effects of known single nucleotide polymorphisms in hTE, some of which have been associated with later-onset loss of structural integrity of elastic tissues and others predicted to affect material properties of elastin matrices on the basis of their location in evolutionarily conserved sites in amniote tropoelastins. Results of these studies show that such sequence variations can significantly alter both the assembly of tropoelastin monomers into a polymeric network and the tensile mechanical properties of that network. Such variations could provide a temporal- or tissue-specific means to customize material properties of elastic tissues to different functional requirements. Conversely, aberrant splicing inappropriate for a tissue or developmental stage or polymorphisms affecting polymeric assembly could compromise the functionality and durability of elastic tissues. To our knowledge, this is the first example of a study that assesses the consequences of known polymorphisms and domain/splice variants in tropoelastin on assembly and detailed elastomeric properties of polymeric elastin.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked ß-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form ß-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties.

Conformational transitions of the cross-linking domains of elastin during self-assembly.

Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that ß-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for ß-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.

Elastin binding protein and FKBP65 modulate in vitro self-assembly of human tropoelastin.

Elastin is a protein that provides the unusual properties of extensibility and elastic recoil to tissues. Assembly of polymeric elastin into its final architecture in the extracellular matrix involves both self-aggregation properties of its monomeric precursor, tropoelastin, and interactions with several matrix-associated proteins that appear to act by modulating the intrinsic self-assembly of tropoelastin. Because of its highly nonpolar character and propensity to self-aggregate, it has been suggested that mechanisms limiting self-aggregation must also be present during the transit of tropoelastin through the cell prior to secretion. Both the elastin binding protein (EBP) and FKBP65 have been suggested to fulfill that role in the Golgi and endoplasmic reticulum compartments of the cell, respectively. However, details about the nature of the interactions between these proteins as well as about the mechanism by which they may act to limit self-aggregation are lacking. In this study, we demonstrate that both EBP and FKBP65 have strong binding affinities for tropoelastin, with the dissociation constant of EBP approximately 4-fold lower than that of FKBP65. Both proteins also modify the kinetics of self-assembly of tropoelastin in an in vitro system, consistent with a role in attenuating the premature intracellular self-aggregation of tropoelastin through a mechanism that limits the growth and maturation of aggregates. The ability of FKBP65 to modulate the self-assembly of tropoelastin is independent of its enzymatic activity to promote the cis-trans isomerization of proline residues in proteins.

Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system.

BACKGROUND: Increasing our understanding of antibiotic resistance mechanisms is critical. To enable progress in this area, methods to rapidly identify and characterize antibiotic resistance conferring enzymes are required. RESULTS: We have constructed a sensitive reporter system in Escherichia coli that can be used to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, resulted in luminescence production as a result of expression of the lux genes carried by the reporter plasmid. Introduction of another plasmid into this system expressing TetX, a tetracycline-inactivating enzyme, caused a marked loss in luminescence due to enzyme-mediated reduction in the intracellular Tc concentration. Data generated for the TetX enzyme using the reporter system could be effectively fit with the known Km and kcat values, demonstrating the usefulness of this system for quantitative analyses. CONCLUSION: Since members of the TetR family of repressors regulate enzymes and pumps acting upon almost every known antibiotic and a wide range of other small molecules, reporter systems with the same design as presented here, but employing heterologous TetR-related proteins, could be developed to measure enzymatic activities against a wide range of antibiotics and other compounds. Thus, the assay described here has far-reaching applicability and could be adapted for high-throughput applications.

A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators.

The tetracycline repressor family transcriptional regulators (TFRs) are homodimeric DNA-binding proteins that generally act as transcriptional repressors. Their DNA-binding activity is allosterically inactivated by the binding of small-molecule ligands. TFRs constitute the third most frequently occurring transcriptional regulator family found in bacteria with more than 10,000 representatives in the nonredundant protein database. In addition, more than 100 unique TFR structures have been solved by X-ray crystallography. In this study, we have used computational and experimental approaches to reveal the variations and conservation present within TFRs. Although TFR structures are very diverse, we were able to identify a conserved central triangle in their ligand-binding domains that forms the foundation of the structure and the framework for the ligand-binding cavity. While the sequences of DNA-binding domains of TFRs are highly conserved across the whole family, the sequences of their ligand-binding domains are so diverse that pairwise sequence similarity is often undetectable. Nevertheless, by analyzing subfamilies of TFRs, we were able to identify distinct regions of conservation in ligand-binding domains that may be important for allostery. To aid in large-scale analyses of TFR function, we have developed a simple and reliable computational approach to predict TFR operator sequences, a temperature melt-based assay to measure DNA binding, and a generic ligand-binding assay that will likely be applicable to most TFRs. Finally, our analysis of TFR structures highlights their flexibility and provides insight into a conserved allosteric mechanism for this family.

The induction of folding cooperativity by ligand binding drives the allosteric response of tetracycline repressor.

Tetracycline (Tc) repressor (TetR) undergoes an allosteric transition upon interaction with the antibiotic, Tc, that abrogates its ability to specifically bind its operator DNA. In this work, by performing equilibrium protein unfolding experiments on wild-type TetR and mutants displaying altered allosteric responses, we have delineated a model to explain TetR allostery. In the absence of Tc, we show that the DNA-binding domains of this homodimeric protein are relatively flexible and unfold independently of the Tc binding/dimerization (TBD) domains. Once Tc is bound, however, the unfolding of the DNA binding domains becomes coupled to the TBD domains, leading to a large increase in DNA-binding domain stability. Noninducible TetR mutants display considerably less interdomain folding cooperativity upon binding to Tc. We conclude that the thermodynamic coupling of the TetR domains caused by Tc binding and the resulting rigidification of the DNA-binding domains into a conformation that is incompatible with DNA binding are the fundamental factors leading to the allosteric response in TetR. This allosteric mechanism can account for properties of the whole TetR family of repressors and may explain the functioning and evolution of other allosteric systems. Our model contrasts with the prevalent view that TetR populates two distinct conformations and that Tc causes a switch between these defined conformations.

Two-way interdomain signal transduction in tetracycline repressor.

The transcription of genes encoding resistance to the antibiotic, tetracycline (Tc), is repressed by tetracycline repressor (TetR), which is a homodimeric alpha-helical protein possessing a small N-terminal DNA binding domain (DNB domain) and a larger C-terminal domain (TBD domain). Binding of Tc to the TBD domain induces a subtle conformational change in the DNB domain that leads to abrogation of its DNA-binding activity, and induction of Tc resistance. While most previous studies on TetR have focused on the effects of Tc-binding on the DNB domain conformation, here we have investigated the role of the DNB domain in modulating Tc binding. We have discovered that a TBD domain construct entirely lacking the DNB domain displays a drastic reduction in Tc-binding affinity even though the DNB domain is far from the Tc-binding site. In the context of full-length TetR, highly destabilizing amino acid substitutions in the DNB domain cause reductions in Tc-binding activity. Strikingly, the DNB domains of these mutants, which are completely unfolded in the absence of Tc, are induced to fold when Tc is bound. These results demonstrate that there is a previously unrecognized two-way interdomain signaling mechanism in TetR whereby the DNB domain is required for maximal Tc-binding by the TBD domain, and Tc-binding in the TBD domain leads to stabilization of the DNB domain. Furthermore, our work suggests that detailed thermodynamic and kinetic studies on mutant forms of other allosteric proteins may also reveal surprising and previously undetected modes of interdomain communication.