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Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA. [ABSTRACT FROM AUTHOR]

Diacylglycerol kinases (DGKs) are multi‐domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+‐triggered activation of DGKα, for which the N‐terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα‐EF induces conformational changes from a protease‐susceptible "open" conformation in the apo state to a well‐folded one in its holo state. Here, we further studied the structural properties of DGKα N‐terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N‐terminal RVH domain is a novel Ca2+‐binding domain, but the Ca2+‐induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten‐globule like structure in the apo state. Further analyses using SEC‐SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca2+, and this interaction is modified in the presence of Ca2+. Taken together, these results present novel insights into the structural rearrangement of DGKα N‐terminal domains upon binding to Ca2+, which is essential for the activation of the enzyme. [ABSTRACT FROM AUTHOR]

Diverse genes associated with familial Parkinson's disease (familial Parkinsonism) have been implicated in mitochondrial quality control. One such gene, PARK7 encodes the protein DJ-1, pathogenic mutations of which trigger its translocation from the cytosol to the mitochondrial matrix. The translocation of steady-state cytosolic proteins like DJ-1 to the mitochondrial matrix upon missense mutations is rare, and the underlying mechanism remains to be elucidated. Here, we show that the protein unfolding associated with various DJ-1 mutations drives its import into the mitochondrial matrix. Increasing the structural stability of these DJ-1 mutants restores cytosolic localization. Mechanistically, we show that a reduction in the structural stability of DJ-1 exposes a cryptic N-terminal mitochondrialtargeting signal (MTS), including Leu10, which promotes DJ-1 import into the mitochondrial matrix for subsequent degradation. Our work describes a novel cellular mechanism for targeting a destabilized cytosolic protein to the mitochondria for degradation. [ABSTRACT FROM AUTHOR]

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer. [ABSTRACT FROM AUTHOR]

Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody-antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol-1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role. [ABSTRACT FROM AUTHOR]

Pore-forming toxins (PFTs) are proteins endowed with metamorphic properties that enable them to stably fold in water solutions as well as in cellular membranes. PFTs produce lytic pores on the plasma membranes of target cells conducive to lesions, playing key roles in the defensive and offensive molecular systems of living organisms. Actinoporins are a family of potent haemolytic toxins produced by sea anemones vigorously studied as a paradigm of a-helical PFTs, in the context of lipid--protein interactions, and in connection with nanopore technologies. We have recently reported that fragaceatoxin C (FraC), an actinoporin, engages biological membranes with a large adhesive motif allowing the simultaneous attachment of up to four lipid molecules prior to pore formation. Since actinoporins also interact with carbohydrates, we sought to understand the molecular and energetic basis of glycan recognition by FraC. By employing structural and biophysical methodologies, we show that FraC engages glycans with low affinity using its lipid-binding module. Contrary to other PFTs requiring separate domains for glycan and lipid recognition, the small single-domain actinoporins economize resources by achieving dual recognition with a single binding module. This mechanism could enhance the recruitment of actinoporins to the surface of target tissues in their marine environment. [ABSTRACT FROM AUTHOR]

Antibodies of the IgG class carry a pair of oligosaccharides (N-glycans) in the Fc region. The importance of the N-glycan is clearly demonstrated by its profound effect in the physicochemical and biological properties of antibodies. The term 'glycoengineering' has been coined to describe contemporary strategies to improve the performance of therapeutic monoclonal antibodies on the basis of modifications in the structure and composition of the N-glycan. These methodologies have resulted in the approval and commercialization of a new generation of antibodies with improved therapeutic efficacy. So far, these advances have been driven by herculean efforts in a process of trial-and-error. The collective work of researchers in this field is progressively revealing the molecular basis of N-glycans for the function of antibodies. This knowledge will ultimately be conducive to the application of rational approaches for the successful manipulation of antibodies using glycoengineering strategies. Herein, we review advances in our understanding of the role of the N-glycan in the structural and dynamic integrity, and biological activity, of antibodies. Since the N-glycan has a multifaceted effect in antibodies, in this review we have emphasized the importance of integrating various techniques that address this problem from multiple points of view. In particular, the combination of X-ray crystallography with nuclear magnetic resonance, molecular dynamics simulations and biophysical approaches based on thermodynamic principles, has emerged as a powerful combination that is deepened our understanding of this unique system with critical implications for human well-being. [ABSTRACT FROM AUTHOR]