United we stand: combining structural methods

Structural biologists benefit enormously by combining structural approaches to tackle biological systems. This is evident in the increasing use of complementary methods combined with the traditional structural biology techniques of macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) and electron microscopy (EM) to generate structural information

Light-responsive self-assembly of a cationic azobenzene surfactant at high concentration.

The formation of high-concentration mesophases by a cationic azobenzene photosurfactant is described for the first time. Using a combination of polarised optical microscopy and small-angle X-ray scattering, optically anisotropic, self-assembled structures with long-range order are reported. The mesophases are disrupted or lost upon UV irradiation

Different modes of interaction by TIAR and HuR with target RNA and DNA

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways

A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant “closed” conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an “open” form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the Kopen value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the Kopen of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg–RKT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes

G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme’s adaptive landscap

X-ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex

The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent- mediated decarboxylation to a dead-end metabolic product

Dynamic Motion and Communication in the Streptococcal C1 Phage Lysin, PlyC

<div><p>The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency.</p></div