Minimal requirements for inhibition of MraY by lysis protein E from bacteriophage ΦX174

The DNA phage ΦX174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides

Capturing the signal

High-resolution structures provide new insights into how an RNA-protein complex recognizes the signal that targets membrane proteins to the endoplasmic reticulum before they aggregate

Animals who think and love : law, identification and the moral psychology of guilt

How does the human animal who thinks and loves relate to criminal justice? This essay takes up the idea of a moral psychology of guilt promoted by Bernard Williams and Herbert Morris. Against modern liberal society’s ‘peculiar’ legal morality of voluntary responsibility (Williams), it pursues Morris’s ethical account of guilt as involving atonement and identification with others. Thinking of guilt in line with Morris, and linking it with the idea of moral psychology, takes the essay to Freud’s metapsychology in Civilization and Its Discontents. Two conflicting routes to guilt are noted in Freud, one involving internalisation of external anger to suppress destructive instincts, the other loving identification with others in the process of self-formation. This second route is developed through the psychoanalytic thought of Hans Loewald and Jonathan Lear. Following Loewald, the moral psychology of self-formation makes loving identification with others the root of responsibility, guilt and atonement. Following Lear, the moral psychology of guilt developed on these lines renders psychoanalysis part of a broadly understood philosophical project following Aristotelian and Socratic principles. Underlying Morris’s account of guilt is the possibility of ‘prospective identification’, understood as the moral and psychological ground of guilt and reconciliation. This is the rational core of criminal justice, which maintains an uneasy relationship with law’s ‘peculiar’ morality

Get5 Carboxyl-terminal Domain Is a Novel Dimerization Motif That Tethers an Extended Get4/Get5 Complex

Tail-anchored trans-membrane proteins are targeted to membranes post-translationally. The proteins Get4 and Get5 form an obligate complex that catalyzes the transfer of tail-anchored proteins destined to the endoplasmic reticulum from Sgt2 to the cytosolic targeting factor Get3. Get5 forms a homodimer mediated by its carboxyl domain. We show here that a conserved motif exists within the carboxyl domain. A high resolution crystal structure and solution NMR structures of this motif reveal a novel and stable helical dimerization domain. We additionally determined a solution NMR structure of a divergent fungal homolog, and comparison of these structures allows annotation of specific stabilizing interactions. Using solution x-ray scattering and the structures of all folded domains, we present a model of the full-length Get4/Get5 complex

A Structural Model of the Sgt2 Protein and Its Interactions with Chaperones and the Get4/Get5 Complex

The insertion of tail-anchored transmembrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2 is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathway via Get4 and Get5. Here, we present the crystal structure from a fungal Sgt2 homolog of the tetratrico-repeat (TPR) domain and part of the linker that connects to the C-terminal domain. The linker extends into the two-carboxylate clamp of the TPR domain from a symmetry-related molecule mimicking the binding to HSCs. Based on this structure, we provide biochemical evidence that the Sgt2 TPR domain has the ability to directly bind multiple HSC family members. The structure allows us to propose features involved in this lower specificity relative to other TPR containing co-chaperones. We further show that a dimer of Sgt2 binds a single Get5 and use small angle x-ray scattering to characterize the domain arrangement of Sgt2 in solution. These results allow us to present a structural model of the Sgt2-Get4/Get5-HSC complex

A large conformational change of the translocation ATPase SecA

The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria. We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-Å resolution. A comparison with the previously determined structures of SecA reveals a nucleotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides. We propose that the open form of SecA represents an activated state

Structures of the Sgt2/SGTA Dimerization Domain with the Get5/UBL4A UBL Domain Reveal an Interaction that Forms a Conserved Dynamic Interface

In the cytoplasm, the correct delivery of membrane proteins is an essential and highly regulated process. The posttranslational targeting of the important tail-anchor membrane (TA) proteins has recently been under intense investigation. A specialized pathway, called the guided entry of TA proteins (GET) pathway in yeast and the transmembrane domain recognition complex (TRC) pathway in vertebrates, recognizes endoplasmic-reticulum-targeted TA proteins and delivers them through a complex series of handoffs. An early step is the formation of a complex between Sgt2/SGTA, a cochaperone with a presumed ubiquitin-like-binding domain (UBD), and Get5/UBL4A, a ubiquitin-like domain (UBL)-containing protein. We structurally characterize this UBD/UBL interaction for both yeast and human proteins. This characterization is supported by biophysical studies that demonstrate that complex formation is mediated by electrostatics, generating an interface that has high-affinity with rapid kinetics. In total, this work provides a refined model of the interplay of Sgt2 homologs in TA targeting

Decoding sequence-level information to predict membrane protein expression

The expression and purification of integral membrane proteins remains a major bottleneck in the characterization of these important proteins. Expression levels are currently unpredictable, which renders the pursuit of these targets challenging and highly inefficient. Evidence demonstrates that small changes in the nucleotide or amino-acid sequence can dramatically affect membrane protein biogenesis; yet these observations have not resulted in generalizable approaches to improve expression. In this study, we develop a data-driven statistical model that predicts membrane protein expression in E. coli directly from sequence. The model, trained on experimental data, combines a set of sequence-derived variables resulting in a score that predicts the likelihood of expression. We test the model against various independent datasets from the literature that contain a variety of scales and experimental outcomes demonstrating that the model significantly enriches expressed proteins. The model is then used to score expression for membrane proteomes and protein families highlighting areas where the model excels. Surprisingly, analysis of the underlying features reveals an importance in nucleotide sequence-derived parameters for expression. This computational model, as illustrated here, can immediately be used to identify favorable targets for characterization