Swarnendu Tripathi

Protein recognition and selection through conformational and mutually induced fit

Significance Protein–protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure is a fascinating problem but not well understood. Calmodulin presents a unique opportunity to investigate mechanisms for selectivity given that it interacts with several hundred different potential targets. In our work, a combined experimental and theoretical approach is taken to define how target selectivity occurs at the molecular level. Our study shows that the binding interactions require mutually induced conformational changes in both calmodulin and the target protein, and broadly informs how intrinsically disordered proteins can achieve both high affinity and high specificity. Protein–protein interactions drive most every biological process, but in many instances the domains mediating recognition are disordered. How specificity in binding is attained in the absence of defined structure contrasts with well-established experimental and theoretical work describing ligand binding to protein. The signaling protein calmodulin presents a unique opportunity to investigate mechanisms for target recognition given that it interacts with several hundred different targets. By advancing coarse-grained computer simulations and experimental techniques, mechanistic insights were gained in defining the pathways leading to recognition and in how target selectivity can be achieved at the molecular level. A model requiring mutually induced conformational changes in both calmodulin and target proteins was necessary and broadly informs how proteins can achieve both high affinity and high specificity.

Conformational frustration in calmodulin–target recognition

Calmodulin (CaM) is a primary calcium (Ca2+)‐signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM‐binding domains from two Ca2+‐CaM‐dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM–CaMKII complex than in the CaM–CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C‐domain of CaM that need to be broken before the formation of intermolecular contacts with the N‐domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations. Copyright

Absence of Activation of DNA Repair Genes and Excellent Efficacy of Phosphaplatins against Human Ovarian Cancers: Implications To Treat Resistant Cancers

Phosphaplatins, platinum(II) and platinum(IV) complexes coordinated to a pyrophosphate moiety, exhibit excellent antitumor activities against a variety of cancers. To determine whether phosphaplatins trigger resistance to treatment by engaging DNA damage repair genes, a yeast genome-wide fitness assay was used. Treatment of yeast cells with pyrodach-2 (D2) or pyrodach-4 (D4) revealed no particular sensitivity to nucleotide excision repair, homologous recombination repair, or postreplication repair when compared with platin control compounds. Also, TNF receptor superfamily member 6 (FAS) protein was overexpressed in phosphaplatin-treated ovarian tumor cells, and platinum colocalized with FAS protein in lipid rafts. An overactivation of sphingomyelinase (ASMase) was noted in the treated cells, indicating participation of an extrinsic apoptotic mechanism due to increased ceramide release. Our results indicate that DNA is not the target of phosphaplatins and accordingly, that phosphaplatins might not cause resistance to treatment. Activation of ASMase and FAS along with the colocalization of platinum with FAS in lipid rafts support an extrinsic apoptotic signaling mechanism that is mediated by phosphaplatins.

Correlation between Gene Variants, Signaling Pathways, and Efficacy of Chemotherapy Drugs against Colon Cancers.

Efficacies, toxicities, and resistance mechanisms of chemotherapy drugs, such as oxaliplatin and 5-fluorouracil (5-FU), vary widely among various categories and subcategories of colon cancers. By understanding the differences in the drug efficacy and resistance at the level of protein–protein networks, we identified the correlation between the drug activity of oxaliplatin/5-FU and gene variations from the US National Cancer Institute-60 human cancer cell lines. The activity of either of these drugs is correlated with specific amino acid variant(s) of KRAS and other genes from the signaling pathways of colon cancer progression. We also discovered that the activity of a non-DNA-binding novel platinum drug, phosphaplatin, is comparable with oxaliplatin and 5-FU when it was tested against colon cancer cell lines. Our strategy that combines the knowledge from pharmacogenomics across cell lines with the molecular information from specific cancer cells is beneficial for predicting the outcome of a possible combination therapy for personalized treatment.

Lessons in Protein Design from Combined Evolution and Conformational Dynamics

Protein-protein interactions play important roles in the control of every cellular process. How natural selection has optimized protein design to produce molecules capable of binding to many partner proteins is a fascinating problem but not well understood. Here, we performed a combinatorial analysis of protein sequence evolution and conformational dynamics to study how calmodulin (CaM), which plays essential roles in calcium signaling pathways, has adapted to bind to a large number of partner proteins. We discovered that amino acid residues in CaM can be partitioned into unique classes according to their degree of evolutionary conservation and local stability. Holistically, categorization of CaM residues into these classes reveals enriched physico-chemical interactions required for binding to diverse targets, balanced against the need to maintain the folding and structural modularity of CaM to achieve its overall function. The sequence-structure-function relationship of CaM provides a concrete example of the general principle of protein design. We have demonstrated the synergy between the fields of molecular evolution and protein biophysics and created a generalizable framework broadly applicable to the study of protein-protein interactions.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca2+-free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng13–49), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng13–49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng13–49 structures by matching the changes in the chemical shifts of CaM upon Ng13–49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski’s equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng13–49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+ release. In contrast, CaMKII peptide increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng13–49 and holoCaM-CaMKII delineates the importance of CaM’s progressive mechanism of target binding on its Ca2+ binding affinities.

Abstract B1-02: Correlation between oncogenic mutations, signaling pathways, and efficacy of platinum-based drugs against colorectal cancers

The purpose of this study is to correlate genetic mutations, amino-acid variants, signaling pathways with platiunum based drug activity to shed light on personalized treatment of cancer. Platinum anticancer drugs such as cisplatin, carboplatin, and oxalipaltin are widely used to treat a variety of cancers. Although DNA is the molecular target for these platinum therapies, their efficacies, toxicities, and resistance mechanisms vary widely among various categories and sub-categories of cancers. To comprehend this great variability, an integrated analysis was performed to determine the impact of somatic mutations on protein functions, signaling pathways, and drug activity (sensitivity or resistance) among the US National Cancer Institute (NCI) 60 human tumor cell lines based on Z-scores to predict a priori treatment outcome using CellMiner (http://discover.nci.nih.gov/cellminer). Specifically, somatic mutations of significantly mutated genes from the cancer genome atlas (TCGA) were analyzed along with the driver genes (oncogenes and tumor suppressor genes) from catalogue of somatic mutations in cancer (COSMIC) for total 188 genes that belong to more than 20 different signaling pathways. The functional impact of individual amino-acid variant for each gene and its correlation with the activity of carboplatin, cisplatin and oxaliplatin for each cancer cell line were explored. Particular attention was given to colon cancer for which nearly 40% of tumors are known to have mutated KRAS (Kirsten rat sarcoma viral oncogene homolog) gene. Resulting analysis revealed that colon cancer cell lines with KRAS mutations for codon 12 (G12V-mutant from SW620 cell line) and codon 13 (G13D-mutant from HCT116 and HCT15 cell lines) correlated with the sensitivity to oxaliplatin. Conversely, oxaliplatin resistant HCC2998 colon cancer cell line did not show any correlation with mutated KRAS for codon 146 (A146T-mutant). Notably, all the colon cancer cell lines were resistant to both carboplatin and cisplatin with no correlation to the KRAS mutants. Based on our integrated analysis we further predicted gene networks related to oxaliplatin activity for colon cancer. The network includes the epidermal growth factor (EGFR) signaling pathway that involves PIK3CA, PIK3CG and MTOR from the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) (PIK3/mTOR) pathway, and JAK3 from Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in addition to KRAS from Mitogen-activated protein kinases (MAPK) pathway. We conclude that instead of considering all mutations of a gene in the same way to assess their clinical significance, it may be beneficial to categorize them into different classes based on their functional impact and efficacies towards the anti-cancer drugs for personalized treatment. Similar analytical approach is being extended to non-small cell lung and ovarian cancers where platinum therapies are widely used. S. Tripathi gratefully acknowledges the support through a training fellowship from a grant by the Cancer Prevention Research Institute of Texas (Grant No. RP140113 to R.N.Bose). Citation Format: Swarnendu Tripathi, Louiza Belkacemi, Margaret S. Cheung, Rathindra N. Bose. Correlation between oncogenic mutations, signaling pathways, and efficacy of platinum-based drugs against colorectal cancers. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-02.