Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex.

The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays.

β-sheet proteins carry out critical functions in biology, and hence are attractive scaffolds for computational protein design. Despite this potential, de novo design of all β-sheet proteins from first principles lags far behind the design of all-α or mixed αβ domains due to their non-local nature and tendency of exposed β-strand edges to aggregate. Through study of loops connecting unpaired β-strands (β-arches), we have identified a series of structural relationships between loop geometry, sidechain directionality and β-strand length that arise from hydrogen bonding and packing constraints on regular β-sheet structures. We use these rules to de novo design jelly-roll structures with double-stranded β-helices formed by 8 antiparallel β-strands. The nuclear magnetic resonance structure of a hyperthermostable design closely matched the computational model, demonstrating accurate control over the β-sheet structure and loop geometry. Our results open the door to the design of a broad range of non-local β-sheet protein structures.

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

Degradation of the non-ionic surfactant polysorbate (PS) has been reported to lead to the formation of visible and subvisible particles that affect product quality. Chemical degradation pathways of PS, such as oxidation and acid/base hydrolysis, have been previously studied, but enzymatic degradation of PS remains poorly understood. In this study, enzyme-mediated hydrolysis of the major components in a heterogeneous mixture of PS20 or PS80 was monitored using an evaporative light scattering detection-high-performance liquid chromatography method. Carboxylester hydrolases from a broad range of organisms were tested, including enzymes from Pseudomonas cepacia, Thermomyces lanuginosus, Candida antarctica, rabbit liver, and pig pancreas. Time course experiments suggested that PS hydrolysis was dependent on the order of esters (mono-, di-, or triester), the identity of the hydrophilic head group (sorbitan or isosorbide), the identity of the fatty acid ester tail (C12 vs C18:1), and the identity of the enzyme. Importantly, no PS component was completely resistant to all the carboxylester hydrolases tested here. Our results illustrate a potential fingerprint approach that could be useful in verifying or identifying enzyme-mediated PS degradation in drug substance and provide an improved understanding of the complexity of PS degradation in the presence of enzymes.