Comment in Structure. 2022 Jun 2;30(6):790-792.

Comment in Structure. 2022 Jun 2;30(6):787-790.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.

Comment on Structure. 2022 Jun 2;30(6):840-850.e6.

Comment on Structure. 2022 Jun 2;30(6):876-885.e5.

Small-angle X-ray scattering (SAXS) experiments are widely used for the characterization of biological macromolecules in solution. SAXS patterns contain information on the size and shape of dissolved particles in nanometer resolution. Here we propose a method for primary SAXS data analysis based on the application of artificial neural networks (NNs). Trained on synthetic SAXS data, the feedforward NNs are able to reliably predict molecular weight and maximum intraparticle distance (Dmax) directly from the experimental data. The method is applicable to data from monodisperse solutions of folded proteins, intrinsically disordered proteins, and nucleic acids. Extensive tests on synthetic SAXS data generated in various angular ranges with varying levels of noise demonstrated a higher accuracy and better robustness of the NN approach compared to the existing methods.

Dynamic allostery emphasizes a role of entropy change manifested as a sole change in protein fluctuations without structural changes. This kind of entropy-driven effect remains largely understudied. The most significant examples involve protein-ligand interactions, leaving protein-protein interactions, which are critical in signaling and other cellular events, largely unexplored. Here we study an example of how protein-protein interaction (binding of Ras to the Ras binding domain [RBD] of the effector protein Raf) affects a subsequent protein association process (Ras dimerization) by quenching Ras internal motions through dynamic allostery. We also investigate the influence of point mutations or ambient temperature, respectively, on the protein dynamics and interaction of two other systems: in adenylate kinase (ADK) and in the EphA2 SAM:Ship2 SAM complex. Based on these examples, we postulate that there are different ways in which dynamic-change-driven protein interactions are manifested and that it is likely a general biological phenomenon.

DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1.

Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. We present a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase β (PI5P4Kβ) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kβ has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kβ use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kβ has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

CD28 has a crucial role in regulating immune responses by enhancing T cell activation and differentiation. Recent studies have shown that the transmembrane helix (TMH) of CD28 mediates receptor assembly and activity, but a structural characterization of TMH is still lacking. Here, we determined the dimeric helix-helix packing of CD28-TMH using nuclear magnetic resonance (NMR) technology. Unexpectedly, wild-type CD28-TMH alone forms stable tetramers in lipid bicelles instead of dimers. The NMR structure of the CD28-TMH C165F mutant reveals that a GxxxA motif, which is highly conserved in many dimeric assemblies, is located at the dimerization interface. Mutating G160 and A164 can disrupt the transmembrane helix assembly and reduces CD28 enhancement in cells. In contrast, a previously proposed YxxxxT motif does not affect the dimerization of full-length CD28, but it does affect CD28 activity. These results imply that the transmembrane domain of CD28 regulates the signaling transduction in a complicated manner.

Nanobodies can achieve remarkable neutralization of genetically diverse pathogens, including HIV-1. To gain insight into their recognition, we determined crystal structures of four llama nanobodies (J3, A12, C8, and D7), all of which targeted the CD4-binding site, in complex with the HIV-1 envelope (Env) gp120 core, and determined a cryoelectron microscopy (cryo-EM) structure of J3 with the Env trimer. Crystal and cryo-EM structures of J3 complexes revealed this nanobody to mimic binding to the prefusion-closed trimer for the primary site of CD4 recognition as well as a secondary quaternary site. In contrast, crystal structures of A12, C8, and D7 with gp120 revealed epitopes that included portions of the gp120 inner domain, inaccessible on the prefusion-closed trimer. Overall, these structures explain the broad and potent neutralization of J3 and limited neutralization of A12, C8, and D7, which utilized binding modes incompatible with the neutralization-targeted prefusion-closed conformation of Env.

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure.