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Additional file 1: Fig. S1. CrkL binds through the SH3N domain and with similar affinity to the four isolated PRMs of C3G. Fig. S2. Analysis of CrkL by sedimentation velocity. Fig. S3. ITC analysis of the binding of CrkL to single-PRM mutants of C3G. Fig. S4. Linear concentration dependence of the nucleotide exchange activity of phospho-C3G. Fig. S5. Binding of CrkL fragments and CrkII to C3G. Table S1. Constructs of human C3G in the vector pETEV15b-His-Halo-TEV for expression in E coli. Table S2. Oligonucleotides used to create constructs of C3G and for site directed mutagenesis. Table S3. Constructs of human C3G in the vectors for expression in mammalian cells. Table S4. Constructs of human CrkL and CrkII in the vectors for expression in E coli. Table S5. Oligonucleotides used to create constructs of CrkL and CrkII. Table S6. Oligonucleotides to subclone the abGFP4 nanobody in a modified pET22b vector. Table S7. Parameters of the CrkL/C3G interaction determined by ITC. Table S8. Hydrodynamic parameters of C3G and CrkL. Table S9. Parameters of the CrkL/C3G interaction determined by ITC and sedimentation velocity. Table S10. Parameters of the CrkL binding to single-PRM mutants of C3G determined by ITC. Table S11. Parameters of the binding of CrkL fragments to C3G determined by ITC.

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This unprecedented mechanism of action resulted in highly potent and selective pathway inhibition, in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this novel mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders.

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum- signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed. Ministry of Science and Innovation of the Spanish Government (PID2019 108778GB C21 (AEI/FEDER, EU)) to WM, (BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU)), to DB, (PID2019-104544GBI00 (AEI/FEDER/EU)) to CA, (PID2019-109320GB-100/AEI/10.13039/501100011033) to SA, (PGC2018-094143-B-I00, (AEI/FEDER, EU)) to FR, and (FIS2016-78313-P) to SA. The Ministry of Science and Innovation of the Spanish Government also funded the Centro Nacional de Biotecnología (CNB) by means of the “Severo Ochoa” Center of Excellence grant SEV 2017-0712. A Wellcome Investigator grant (209500) to Jeff Errington supported LW. Institutional grants from the ‘Fundación Ramón Areces’ and ‘Banco de Santander’ supported

Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. Rap is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by Rco in the absence of the mature pLS20 signaling peptide Phr*. We report here an in-depth structural study of apo and Phr*-bound states of Rap at various levels of atomic detail. We show that apo-Rap is dimeric and that Phr*-bound Rap forms NTD-mediated tetramers. In addition, we show that Rap binds Rco directly in the absence of Phr* and that addition of Phr* releases Rco from Rap. This allows Rco to bind the promotor region of crucial conjugation genes blocking their expression.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.