Tightly bound conserved water in Pin1 maintains critical hydrogen-bond network.
Weaker bound exchangeable water is present in a distinctive helix-turn-coil motif.
CD spectra confirm the requirement of protein environment to stabilize the motif. 4.3 Introduction
Water molecules play a significant role in chemical biology and have direct involvement with biomolecules, especially when they are localized in well-defined pockets. They are crucial for biomolecular stability, flexibility and function (Nagendra et al., 1998). The presence of water at an internal cavity stabilizes protein structures by hydrating the polar atoms, and the loss of these localized internal water molecules can induce structural unfolding of protein structures (Robinson and Cho, 1999). Conserved water molecules have been suggested to play important roles in stabilization of loop structure for protein-protein interactions, such as the Ω loop in the class A β-lactamase (Bös and Pleiss, 2008). Structurally, conserved water molecules have been found to be an integral part of several classes of proteins, including ribonuclease T1 (Loris et al., 1999), serine proteases (Sreenivasan and Axelsen, 1992), Rossmann fold dinucleotide binding proteins (Bottoms et al., 2002), MHC class 1 proteins (Ogata and Wodak, 2002), aspartic proteinases (Prasad and Suguna, 2002), and protein kinases (Knight et al., 2009). Individual water molecules can be crucial in organic reactions and enzyme function (Kahne and Still, 1988). Water molecules actively participate in hydrolysis and esterification reactions and play a major role in chemical catalysis by taking part in transition state stabilization (Kahne and Still, 1988;Romero and de Meis, 1989). Moreover, studies indicate that water molecules can be crucial for protein-ligand recognition by bridging the interactions (Hamelberg and McCammon, 2004;Sahai and Biggin, 2011). Bridging the interactions of water with ligands can be exploited to enhance the affinity of ligands in drug discovery processes (Michel et al., 2009;Gianti et al., 2015). Another important role of water includes regulating the dynamics of proteins (Hamelberg et al., 2006;Johnson et al., 2010). Conserved water molecules at a site distal from the active site
have been identified in several families of proteins to play significant roles in maintaining the structural integrity of the active site (Barycki et al., 2001). Water molecules can also act as allosteric modulators that can alter the dynamics and function of biomolecules (Prakash et al., 2012). Usually, these structural waters are conserved within a protein family, that is, they occupy nearly identical three-dimensional locations with reference to their associated structures to perform the same function. Recent computational and experimental studies show that mutating the critical water-interacting residue can disrupt the interaction network and alter enzyme activity (Szep et al., 2009;Park and Saven, 2005). These studies therefore indicate that structural water molecules located within the protein core can play an important role, modulating protein structure, flexibility, ligand binding and function.
Peptidyl proline isomerases (PPIases) are a family of enzymes that catalyzes cis/trans isomerizations of peptide bonds preceding a proline residue (Lang et al., 1987;Yaffe et al., 1997). Pin1 belongs to the Parvulin family of PPIases and catalyzes cis/trans isomerizations of the prolyl ω bond when the preceding residue is a phosphorylated Ser (pSer) or Thr (pThr) (Bayer et al., 2003;Yaffe et al., 1997;Schutkowski et al., 1998;Ranganathan et al., 1997). Pin1 controls the regulatory action of a pool of cis/trans pSer/pThr motifs in a multitude of cellular signaling pathways, including DNA repair, cell growth and division, apoptosis and transcription (Lu and Zhou, 2007). The synergy between posttranslational phosphorylation and catalysis of cis/trans isomerizations by Pin1 has implications for the proper function of many sub-cellular processes in order to prevent diseases, involving cancers (Theuerkorn et al., 2011;Lu and Zhou, 2007) and Alzheimer’s (Bulbarelli et al., 2009;Lu et al., 1999;Pastorino et al., 2006). Pin1 is therefore a potential therapeutic target for the treatment of many life-threatening diseases. Structurally, Pin1 contains two conserved water molecules, henceforth referred to as Wat1 and
Wat2, near the active site (Figure 1A). Wat1 forms a hydrogen-bonding network with residues His59, Ser111 and Ser115 (Figure 1B). His59 and Ser115 interact with a critical active site Cys113 residue, which is important for the function and regulation of Pin1 (Ranganathan et al., 1997;Barman and Hamelberg, 2014;Xu et al., 2014;Wang et al., 2015). Wat2 is located near the substrate binding site and is within the crevice of a helix-turn-coil structural motif, which is akin to a well-known EF-hand localization motif for calcium ions (Nakayama and Kretsinger, 1994). Wat2 forms a hydrogen-bonding network with Gln129, Met130 and Glu135 (Figure 1C).
Figure 4.1 Conserved water binding in Pin1.
A) Location of the two conserved water molecules Wat1 and Wat2 in the Pin1 crystal structure (PDB id: 2Q5A). B) Interaction of Wat1 with neighboring active site residues. C) Interaction of Wat2 with the surrounding residues. The helix-turn-coil motif is shown in yellow cartoon. D) The crystallographic B-factors of Wat1 (black) and Wat2 (red) in the corresponding X-ray structure.
Studies are currently being carried out to investigate the atomistic detail of the catalytic mechanism of Pin1 through a number of experimental and theoretical studies (Greenwood et al.,
2011;Velazquez and Hamelberg, 2011;Velazquez and Hamelberg, 2013;Vöhringer-Martinez et al., 2012;Schutkowski et al., 1998;Barman and Hamelberg, 2014;Ranganathan et al., 1997;Mercedes-Camacho et al., 2013;Namanja et al., 2011;Di Martino et al., 2014). However, the significance of these conserved water molecules has never been addressed to the best of our knowledge. In this study, detailed free energy calculations and MD simulations have been utilized to address structural and functional roles of these two water molecules. Furthermore, CD spectroscopic studies were performed on the isolated helix-turn-coil motif consisting of 20 amino acid residues to investigate its stability and isolated conformational ensemble. A structural bioinformatics study was also undertaken to investigate the prevalence of similar structural motifs in a non-redundant protein database. Additional atomistic details of Pin1 and its localized hydration may provide necessary information on fully understanding the mechanism of Pin1. A unique structural motif in proteins that can trap water molecules has also been revealed. The motif can serve as an elementary unit in protein engineering and enzyme design.