Structure, Oligomerisation and Interactions of β-Lactoglobulin

β-Lactoglobulin (βLg), as the most abundant whey protein in ruminant milk and as a useful model protein, is the subject of countless biophysical studies in the literature, yet its physiological role is hitherto unknown. This chapter deals with studies that focus on the structure of βLg, its oligomeric behaviour and the interactions that this protein participates in. These and further studies are necessary to understand how the protein’s physicochemical properties may influence the processing, digestion and immunogenicity of ruminant milks and their products. However, there is also a need for research into the interactions that occur naturally between βLg and other components in milk, as this may give us insight into the physiological role of the protein

Structural Basis of the Autophagy-Related LC3/Atg13 LIR Complex: Recognition and Interaction Mechanism

SummaryAutophagy is a bulk degradation pathway that removes cytosolic materials to maintain cellular homeostasis. The autophagy-related gene 13 (Atg13) and microtubule associate protein 1 light chain 3 (LC3) proteins are required for autophagosome formation. We demonstrate that each of the human LC3 isoforms (LC3A, LC3B, and LC3C) interacts with Atg13 via the LC3 interacting region (LIR) of Atg13. Using X-ray crystallography, we solved the macromolecular structures of LC3A and LC3C, along with the complex structures of the LC3 isoforms with the Atg13 LIR. Together, our structural and binding analyses reveal that the side-chain of Lys49 of LC3 acts as a gatekeeper to regulate binding of the LIR. We verified this observation by mutation of Lys49 in LC3A, which significantly reduces LC3A positive puncta formation in cultured cells. Our results suggest that specific affinity of the LC3 isoforms to the Atg13 LIR is required for proper autophagosome formation

Microfluidics for Small-Angle X-ray Scattering

Small-angle X-ray scattering is a well-established biophysical technique, whilst micro-fluidics is proving to be a convenient technology for creating miniaturised multifunctional devices. Both fields are highly versatile and find use in multiple scientific disciplines. Together, they offer the potential to obtain structural information on biomacromolecules, nanoparticles and condensed matter, in a high-throughput manner and with enhanced time-resolution capabilities. This chapter provides practical design considerations for X-ray-based microfluidic systems and examines some of the existing microfluidic platforms used in conjunction with small-angle X-ray scattering. As the exclusive advantages of microfluidics become recognised and accessible, the prevalence of microfluidic sample environments in X-ray scattering measurements will hopefully increase

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PKTIM+Reg) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PKTIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PKTIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes

Structural and Functional analysis of the GABARAP interaction motif (GIM)

© 2017 The Authors. Published under the terms of the CC BY 4.0 license. Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X 1 -X 2 -[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F] -[V/I]-X 2 -V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions

L,L-diaminopimelate aminotransferase (DapL): a putative target for the development of narrow-spectrum antibacterial compounds.

Despite the urgent need for sustained development of novel antibacterial compounds to combat the drastic rise in antibiotic resistant and emerging bacterial infections, only a few clinically relevant antibacterial drugs have been recently developed. One of the bottlenecks impeding the development of novel antibacterial compounds is the identification of new enzymatictargets.Thenutritionallyessentialaminoacidanabolicpathways,forexamplelysinebiosynthesis,provideanopportunitytoexplorethedevelopmentofantibacterialcompounds,sincehumangenomesdonotpossessthegenesnecessarytosynthesizetheseaminoacidsdenovo.Thediaminopimelate(DAP)/lysine(lys)anabolicpathwaysareattractivetargetsforantibacterialdevelopmentsincethepenultimatelysprecursormeso-DAP(m-DAP)isacross-linkingaminoacidinthepeptidoglycan(PG)cellwallofmostGram-negativebacteriaandlysplaysasimilarroleinthePGofmostGram-positivebacteria,inadditiontoitsroleasoneofthe20proteogenicaminoacids.TheL,L-diaminopimelateaminotransferase(DapL)pathwaywasrecentlyidentifiedasanovelvariantoftheDAP/lysanabolicpathways.TheDapLpathwayhasbeenidentifiedinthepathogenicbacteriabelongingtothegenus;Chlamydia,Leptospira,andTreponema.ThedapLgenehasbeenidentifiedinthegenomesof381orapproximately13%ofthe2771bacteriathathavebeensequenced,annotatedandrepositedintheNCBIdatabase,asofMay23,2014.ThenarrowdistributionoftheDapLpathwayinthebacterialdomainprovidesanopportunityforthedevelopmentandordiscoveryofnarrowspectrumantibacterialcompounds

DNA storage—from natural biology to synthetic biology

Natural DNA storage allows cellular differentiation, evolution, the growth of our children and controls all our ecosystems. Here, we discuss the fundamental aspects of DNA storage and recent advances in this field, with special emphasis on natural processes and solutions that can be exploited. We point out new ways of efficient DNA and nucleotide storage that are inspired by nature. Within a few years DNA-based information storage may become an attractive and natural complementation to current electronic data storage systems. We discuss rapid and directed access (e.g. DNA elements such as promotors, enhancers), regulatory signals and modulation (e.g. lncRNA) as well as integrated high-density storage and processing modules (e.g. chromosomal territories). There is pragmatic DNA storage for use in biotechnology and human genetics. We examine DNA storage as an approach for synthetic biology (e.g. light-controlled nucleotide processing enzymes). The natural polymers of DNA and RNA offer much for direct storage operations (read-in, read-out, access control). The inbuilt parallelism (many molecules at many places working at the same time) is important for fast processing of information. Using biology concepts from chromosomal storage, nucleic acid processing as well as polymer material sciences such as electronical effects in enzymes, graphene, nanocellulose up to DNA macramé , DNA wires and DNA-based aptamer field effect transistors will open up new applications gradually replacing classical information storage methods in ever more areas over time (decades)

Genomic and biochemical analysis of the diaminopimelate and lysine biosynthesis pathway in Verrucomicrobium spinosum: Identification and partial characterization of L,L-diaminopimelate aminotransferase and UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate ligase

The Gram-negative bacterium Verrucomicrobium spinosum has attracted interest in recent years following the sequencing and annotation of its genome. Comparative genomic analysis of V. spinosum using diaminopimelate/lysine metabolic genes from Chlamydia trachomatis suggests that V. spinosum employs the L,L-diaminopimelate aminotransferase (DapL) pathway for diaminopimelate/lysine biosynthesis. The open reading frame corresponding to the putative dapL ortholog was cloned and the recombinant enzyme was shown to possess L,L-diaminopimelate aminotransferase activity in vitro. In vivo analysis using functional complementation confirmed that the dapL ortholog was able to functionally complement an E. coli mutant that confers auxotrophy for diaminopimelate and lysine. In addition to its role in lysine biosynthesis, the intermediate diaminopimelate has an integral role in peptidoglycan biosynthesis. To this end, the UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-meso-diaminopimelate ligase ortholog was also identified, cloned and was shown to possess meso-diaminopimelate ligase activity in vivo. The L,L-diaminopimelate aminotransferase pathway has been experimentally confirmed in several bacteria, some of which are deemed pathogenic to animals. Since animals, and particularly humans, lack the genetic machinery for the synthesis of diaminopimelate/lysine de novo, the enzymes involved in this pathway are attractive targets for development of antibiotics. Whether dapL is an essential gene in any bacteria is currently not known. V. spinosum is an excellent candidate to investigate the essentiality of dapL, since the bacterium employs the DapL pathway for lysine and cell wall biosynthesis, is non-pathogenic to humans, facile to grow and can be genetically manipulated

The fitness challenge of studying molecular adaptation

Advances in bioinformatics and high-throughput genetic analysis increasingly allow us to predict the genetic basis of adaptive traits. These predictions can be tested and confirmed, but the molecular-level changes—i.e. the molecular adaptation—that link genetic differences to organism fitness remain generally unknown. In recent years, a series of studies have started to unpick the mechanisms of adaptation at the molecular level. In particular, this work has examined how changes in protein function, activity, and regulation cause improved organismal fitness. Key to addressing molecular adaptations is identifying systems and designing experiments that integrate changes in the genome, protein chemistry (molecular phenotype), and fitness. Knowledge of the molecular changes underpinning adaptations allow new insight into the constraints on, and repeatability of adaptations, and of the basis of non-additive interactions between adaptive mutations. Here we critically discuss a series of studies that examine the molecular-level adaptations that connect genetic changes and fitnes