During the fiscal year, we accomplished the following: 1. Using all atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 insert loop was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis. 2. PI3K (Phosphatidylinositol 3-kinase ) is a key lipid kinase in the PI3K/Akt pathway. Its frequent oncogenic mutations make it a primary drug target. Calmodulin (CaM) activates PI3K independently of extracellular signals, indicating a significant role in oncogenic PI3K activation. Here, we reveal the atomic-scale structures of CaM in complexes with the nSH2 and cSH2 domains of the regulatory p85 subunit of PI3K, and illustrate how CaM activates PI3K by targeting the soft 1-5-10 CaM-binding motifs in both the nSH2 and cSH2 domains of p85. Experimental data reveal that CaM binds cSH2 first, followed by nSH2 binding hours later. CaM typically prefers binding helical peptides. Here we observe that unlike in cSH2, the CaM-binding motif in nSH2 populates a mixed -sheet/-helix/random coil structure. The population shift from a -sheet toward CaMs favored -helical conformation explains why the nSH2 domain needs a longer time for CaM binding in the experiments. The soft CaM-binding motifs in both nSH2 and cSH2 domains establish strong CaM-PI3K interactions, collectively facilitating PI3K activation. This work uncovers the structural basis for CaM-driven PI3K activation. 3. Developed an approach using tandem mass spectrometry (MS/MS) to rapidly identify and classify multiple microorganisms. Rapid and accurate identification of microorganisms is important for public health and safety. We demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. The proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available to interested investigators.

Agency
National Institute of Health (NIH)
Institute
Clinical Center (CLC)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIACL080017-08
Application #
9785974
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Clinical Center
Department
Type
DUNS #
City
State
Country
Zip Code
Ozdemir, E Sila; Jang, Hyunbum; Gursoy, Attila et al. (2018) Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2. J Biol Chem 293:3685-3699
Zhang, Mingzhen; Li, Zhigang; Wang, Guanqiao et al. (2018) Calmodulin (CaM) Activates PI3K? by Targeting the ""Soft"" CaM-Binding Motifs in Both the nSH2 and cSH2 Domains of p85?. J Phys Chem B :
Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y et al. (2018) Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry. J Am Soc Mass Spectrom 29:1721-1737
Li, Zhigang; Zhang, Yonghong; Hedman, Andrew C et al. (2017) Calmodulin Lobes Facilitate Dimerization and Activation of Estrogen Receptor-?. J Biol Chem 292:4614-4622
Chawla, Bhavna; Hedman, Andrew C; Sayedyahossein, Samar et al. (2017) Absence of IQGAP1 Protein Leads to Insulin Resistance. J Biol Chem 292:3273-3289
LeCour Jr, Louis; Boyapati, Vamsi K; Liu, Jing et al. (2016) The Structural Basis for Cdc42-Induced Dimerization of IQGAPs. Structure 24:1499-508
Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C et al. (2016) IQGAP1 Binds To YAP and Modulates Its Transcriptional Activity. J Biol Chem :
Choi, Suyong; Hedman, Andrew C; Sayedyahossein, Samar et al. (2016) Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases. Nat Cell Biol 18:1324-1335
Bessède, Emilie; Molina, Silvia; Acuña-Amador, Luis et al. (2016) Deletion of IQGAP1 promotes Helicobacter pylori-induced gastric dysplasia in mice and acquisition of cancer stem cell properties in vitro. Oncotarget 7:80688-80699
Byrnes, Colleen; Lee, Y Terry; Meier, Emily R et al. (2016) Iron dose-dependent differentiation and enucleation of human erythroblasts in serum-free medium. J Tissue Eng Regen Med 10:E84-9

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