A protein coupling and molecular simulation analysis of the clinical mutants of androgen receptor revealed a higher binding for Leupaxin, to increase the prostate cancer invasion and motility

Highlights

• Leupaxin (LPXN) interact with Androgen receptor (AR) and play a significant role in the invasion and progression of PCa.

• However, no detailed study is available on how the LPXN interacts with AR and increases the prostate cancer progression.

• The current study also uses state-of-the-art methods to provide atomic-level insights into the binding of AR and LPXN.

• Protein coupling analysis revealed that the three mutants favour the robust binding of LPXN than the wild type.

• AR-LPXN in prostate cancer is important to decipher the molecular mechanism of disease and therapeutics developments.

Abstract

Recently a novel coactivator, Leupaxin (LPXN), has been reported to interact with Androgen receptor (AR) and play a significant role in the invasion and progression of prostate cancer. The interaction between AR and LPXN occurs in a ligand-dependent manner and has been reported that the LIM domain in the Leupaxin interacts with the LDB (ligand-binding domain) domain AR. However, no detailed study is available on how the LPXN interacts with AR and increases the (prostate cancer) PCa progression. Considering the importance of the novel co-activator, LPXN, the current study also uses state-of-the-art methods to provide atomic-level insights into the binding of AR and LPXN and the impact of the most frequent clinical mutations H874Y, T877A, and T877S on the binding and function of LPXN. Protein coupling analysis revealed that the three mutants favour the robust binding of LPXN than the wild type by altering the hydrogen bonding network. Further understanding of the binding variations was explored through dissociation constant prediction which demonstrated similar reports as the docking results. A molecular simulation approaches further revealed the dynamic features which reported variations in the dynamics stability, protein packing, hydrogen bonding network, and residues flexibility index. Furthermore, we also assessed the protein motion and free energy landscape which also demonstrated variations in the internal dynamics. The binding free energy calculation revealed -32.95 ± 0.17 kcal/mol for the wild type, for H874Y the total binding energy (BFE) was -36.69 ± 0.11

kcal/mol, for T877A the BFE was calculated to be -38.78 ± 0.17 kcal/mol while for T877S the BFE -41.16 ± 0.12 kcal/mol. This shows that the binding of LPXN is increased by these mutations which consequently increase the PCa invasion and motility. In conclusion, the current study helps in understanding the protein networks and particular the coupling of AR-LPXN in prostate cancer and is of great interest in deciphering the molecular mechanism of disease and therapeutics developments.

Introduction

Prostate cancer (PCa), the malignant growth of tumors in prostate glands, is a widely spread cancer in industrialized countries. In men, it is ranked as the second leading cause of mortality. In the United States (USA) after skin cancer, PCa is considered as the most common type of cancer and largely remains asymptomatic [1]. Due to the slow-growing rate, PCa is not that much deadly but some aggressive tumors in the prostate glands can cause serious problems and consequently deaths. PCa first develops as an androgen-dependent lesion that can be efficaciously treated upon surgical removal. However, targeting the androgen receptor (AR) by using an androgen antagonist successfully increased the survival rate and overall treatment [2]. Initially, the AR-ablation treatment responded in 80% of the patients however later on the treatment efficacy was reduced due to the reports of novel mutations in the AR which cause resistance to various agonists [3,4]. The four regions of AR include N-terminal, an NH2 terminal transactivation domain (NTD), a DNA-binding domain (DBD), a hinge region, and a ligand-binding domain (LBD). The LBD has been reported to have many mutations responsible for resistance to various drugs [5,6]. Among the reported mutations L702H, W741C, H874Y, F876L, T877A, T877S, and T878A are included while the T877A, T877S, and T878A are the most frequently reported mutations [7,8].

The coupling of AR with co-activator proteins helps in the development of tumors that are insensitive to AR therapy consequently altering the effectiveness of AR-based treatments of PCa. Cellular protein networks and protein coordination are essential to any type of cancer and therapeutics evasion [9,10]. For instance, the interaction between AR and Leupaxin (LPXN) has been reported to play a significant role in the PCa invasion and progression. LPXN is a transcriptional coactivator for AR and helps in cell adhesion, transcription regulation, and cell migration in PCa. The interaction between AR and LPXN occurs in a ligand-dependent manner. A yeast two-hybrid experiments-based investigation revealed that the LIM domain in the Leupaxin interacts with the AR. In this regard, a conserved motif “FXXLF” corresponds to position 380–384 at the N-terminal of LPXN and was observed to be responsible for coupling with AR. Alanine substitutions “AXXAA” results in a significant decline in the binding between LPXN and AR. Moreover, the LIM 1 and LIM 2 domains are not responsible for interaction with the AR [9]. On the other hand, the ligand-binding domain (LBD) corresponds to position 669–920 and is responsible for the interaction with LPXN and other proteins. The AR receptor has been reported to be involved in N/C (amino/carboxyl-terminal) interaction pattern for co-regulator recruitment during transcriptional activation. The interaction between AR and LPXN has been responsible for transcriptional activation of AR in PCa and significant morphological and biochemical variations were observed when RNA interference was used. Moreover complete knockdown of LPXN reported a loss of invasive and motility in the PCa cells [9]. The study concluded that LPXN acts as a novel coactivator for AR and thus acts as a potential therapeutic biomarker for the treatment of PCa.

Understanding the protein networks and particular the coupling of two or more proteins in a complex disease like cancer is of great interest to decipher the molecular mechanism of disease and therapeutics developments [11]. In this regard computational methods such as protein-protein coupling, molecular modelling and simulation provide a better view of such associations [[12], [13], [14]]. Previously these approaches have been used to explore the mutational landscape of different proteins such as pyrazinamide, estrogen receptor, ERBB3, and their role in various diseases [[15], [16], [17], [18], [19], [20]]. Considering the importance of the novel co-activator, LPXN, the current study also uses state-of-the-art methods to provide atomic-level insights into the binding of AR and LPXN. The three clinical mutations H874Y, T877A, and T877S are reported to be most frequently reported in most of the PCa patients. Thus herein, the protein complexes were predicted for AR and LPXN of the wild type, and H874Y, T877A, and T877S mutants further comprehend the role of these mutations on the binding of AR and its co-activator, LPXN. Using HADDOCK we performed protein-protein docking to determine the binding variations between the wild type and H874Y, T877A, and T877S mutants. We further used molecular simulation approaches to understand the dynamic features such as stability, flexibility, the radius of gyration, and hydrogen bonding patterns. In addition, the protein motions in each complex followed by a free energy landscape were performed for further understanding of the binding of AR and its co-activator, LPXN. Finally, the free energy calculation methods i.e. MM/GBSA approach was used to demonstrate the binding variations between the wild type and H874Y, T877A, and T877S. In conclusion, the current atomic level understanding would help to provide knowledge on the key hotspot for the therapeutics development for the treatment of PCa.