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Single-molecule studies on PolySUMO proteins reveal their mechanical flexibility.

ABSTRACT: Proteins with ?-sandwich and ?-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (?130 pN) and SUMO2 (?120 pN) are lower than that of ubiquitin (?190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (?0.51 nm) and SUMO2 (?0.33 nm) are much larger than that of ubiquitin (?0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.

SUBMITTER: Kotamarthi HC

PROVIDER: S-EPMC3660650 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Single-molecule studies on PolySUMO proteins reveal their mechanical flexibility.

Kotamarthi Hema Chandra HC Sharma Riddhi R Koti Ainavarapu Sri Rama SR

Biophysical journal 20130501 10

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-l ...[more]

PMID: 23708367

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Project description:Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, whereas PKCα is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

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Single-molecule studies on PolySUMO proteins reveal their mechanical flexibility.

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Single-molecule studies on PolySUMO proteins reveal their mechanical flexibility.

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