OVID-19 is caused by the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the majority of COVID-19 infections are relatively mild, with recovery typically within 2-3 weeks 1,2 , a significant number of patients develop severe illness, which is postulated to be related to both an overactive immune response and viral-induced pathology 3,4. The role of T cell immune responses in disease pathogenesis and longer-term protective immunity is currently poorly defined, but essential to understand in order to inform therapeutic interventions and vaccine design. Currently, there are many ongoing vaccine trials, but it is unknown whether they will provide long-lasting protective immunity. Most vaccines are designed to induce antibodies to the SARS-CoV-2 spike protein, but it is not yet known if this will be sufficient to induce full protective immunity to SARS-CoV-2 (refs. 5-8). Studying natural immunity to the virus, including the role of SARS-CoV-2specific T cells, is critical to fill the current knowledge gaps for improved vaccine design. For many primary virus infections, it typically takes 7-10 d to prime and expand adaptive T cell immune responses in order to control the virus 9. This coincides with the typical time it takes for patients with COVID-19 to either recover or develop severe illness. There is an incubation time of 4-7 d before symptom onset and a further 7-10 d before individuals progress to severe disease 10 .

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal-DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a 'mexican wave' of positional and conformational changes propagating sequentially along this belt.

The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole. The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52. It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by beta-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein-base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 A. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

Summary By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

Bacillus subtilis uses novel regulatory mechanisms in controlling expression of its genes of tryptophan synthesis and transport. These mechanisms respond to changes in the intracellular concentrations of free tryptophan and uncharged tRNA(Trp). The major B. subtilis protein that regulates tryptophan biosynthesis is the tryptophan-activated RNA-binding attenuation protein, TRAP. TRAP is a ring-shaped molecule composed of 11 identical subunits. Active TRAP binds to unique RNA segments containing multiple trinucleotide (NAG) repeats. Binding regulates both transcription termination and translation in the trp operon, and translation of other coding regions relevant to tryptophan metabolism. When there is a deficiency of charged tRNA(Trp), B. subtilis forms an anti-TRAP protein, AT. AT antagonizes TRAP function, thereby increasing expression of all the genes regulated by TRAP. Thus B. subtilis and Escherichia coli respond to identical regulatory signals, tryptophan and uncharged tRNA(Trp), yet they employ different mechanisms in regulating trp gene expression.

Tyrosine phenol-lyase (EC 4.1.99.2) from Citrobacter freundii has been cloned and the primary sequence deduced from the DNA sequence. From the BrCN digest of the NaBH4-reduced holoenzyme, five peptides were purified and sequenced. The amino acid sequences of the peptides agreed with the corresponding parts of the tyrosine phenol-lyase sequence obtained from the gene structure. K257 is the pyridoxal 5'-phosphate binding residue. Assisted by the sequence data, the crystal structure of apotyrosine phenol-lyase, a pyridoxal 5'-phosphate-dependent enzyme, has been refined to an R-factor of 16.2% at 2.3-%L resolution using synchrotron radiation diffraction data. The tetrameric molecule has 222 symmetry, with one of the axes coincident with the cr stallographic 2-fold symmetry axis of the crystal which belongs to the space group P2,212 with a = 76.0 l, b = 138.3 A, and c = 93.5 A. Each subunit comprises 14 a-helices and 16 &strands, which fold into a small and a large domain. The coenzyme-binding lysine residue is located at the interface between the large and small domains of one subunit and the large domain of a crystallographically related subunit. The fold of the large, pyridoxal 5'-phosphate binding domain and the location of the active site are similar to that found in aminotransferases. Most of the residues which participate in binding of pyridoxal 5'-phosphate in aminotransferases are conserved in the structure of tyrosine phenol-lyase. Two dimers of tyrosine phenol-lyase, each of which has a domain architecture similar to that found in aspartate aminotransferases, are bound together through a hydrophobic cluster in the center of the molecule and intertwined N-terminal arms.