Role of highly central residues of P-loop and it's flanking region in preserving the archetypal conformation of Walker A motif of diverse P-loop NTPases

P-loop NTPases represent a large and highly diverse protein family that is involved in variety of cellular functions. Walker A motif forms a typical arched conformation, necessary to accommodate the phosphate moiety of the nucleoside tri (or di-) phosphate in Ploop NTPases. The feature that maintains the ancient architecture of P-loop is unidentified and uncharacterized. Here, using a well established global network parameter, closeness centrality, we identify that Walker A and its flanking regions (N- and C-terminal) have high density of globally connected residue positions. We find that closeness centrality of these residue positions are conserved across common structural core of diverse domains of P-loop NTPase fold. Our results suggest the potential role of globally connected residues in maintaining the local conformation of P-loop.

residue positions in P-loop NTPases. We report that closeness centrality of these residue positions are conserved across common structural core of Ras superfamily and diverse domains of P-loop NTPase fold. No such high densities of high centrality residue positions are observed in the proteins containing Walker A sequence that do not form P-loop. The presented data clearly indicate the role of globally connected residues in conservation of the local conformation of an ancient motif such as Walker A.

Methodology: Selection of structures of P-loop containing NTPases
High resolution X-ray crystallographic structures of diverse domain of P-loop containing NTPases were used in the study. Initially, ScopTree search of protein databank (http://www.rcsb.org/pdb) was used to retrieve a set of 1203 structures of P-loop containing nucleoside triphosphate hydrolase. The search was then refined to 227 distantly related protein structures by using ScopTree homologue removal tool at 30% sequence identity cutoff. This was primarily done to avoid redundancy and utilize the diversity present in the P loop NTPases. Complete structures (i.e., without chain breaks or missing residues) with resolution ≤ 2.4 were chosen. Finally, we selected 23 structures of P-loop NTPases Table 1 (see supplementary material). We retrieved 22 PDB files for protein structures containing Walker A sequence (GXXXXGKS/T) that do not form the P-loop Table2 (see supplementary material) [7].

Computation of closeness centrality
Protein structures can be represented as a residue-residue interaction graphs in which amino acid residues serve as the nodes and their interatomic contacts are the edges. Closeness centrality correlates more accurately with critical residues than any other centrality measurement tested [12]. Therefore, we used SARIG server which efficiently calculates the closeness centrality (please see supplementary material for calculation and explanation).
Beginning with the atomic coordinates of a protein structure, server calculates the interaction between each pair of atoms by using the CSU program [14]. Closeness values were calculated for each residue and standardized by calculating their standard deviation from the mean value. The z-score of the closeness centrality was calculated by z-score = (C (x) − μ) / σ, where µ is the mean value of closeness and σ is the standard deviation. The residues with z-score ≥ 1.0 were considered significant (for detailed descriptions, please refer to Amitai et al [8]). Protein structure analysis was performed using Chimera (http://plato.cgl.ucsf.edu/chimera).

Results and Discussion:
Walker A motif forms a typical architecture in P-loop fold NTPase (Figure1A & 1B). A distortion in the P-loop conformation makes it incompatible with the binding of nucleotides [15]. The features that contribute in preserving the architecture of this ancient motif remain unidentified and uncharacterized. Therefore, an important and open question is how P-loop forms a typical architecture in structurally and functionally diverse P-loop NTPases. Here, we used a well established closeness centrality network parameter to study the global impact of residues on the typical local conformation of Ploop. Residues with high closeness value are central in network and interact with other residues directly or by a few intermediates [8].

High closeness residue positions around P-loop and its flanking regions in Ras Super family members
In order to understand the P-loop architecture, we first analyzed the residue-residue interaction network of Ras superfamily (Ras: 5P21; Rab: 3RAB; Ran: 1IBR, Rho: 1M7B and Arf: 1R4A) experimental structures in GTP bound form. Interestingly, Walker A and its flanking regions showed high density of high closeness residue positions (Table 1). Here, the high closeness centrality positions are defined as those positions with statistically significant closeness values (z-score ≥1.0). Five Walker A residue positions (W1, W2, W5, W6, W7), four contiguous N-terminal residue positions (N2-N5) and two Cterminal residues (C2 and C3), flanking the Walker A, showed high closeness centrality in Ras superfamily members (Table 1).

High density of high closeness residue positions in P-loop and its flanking regions in diverse set of P-loop NTPases
Since the Ras superfamily belongs to P-loop NTPase fold, we then extended the centrality analysis on high resolution X-ray crystallographic structures of P-loop NTPases ( Table 1). The structural overlay of highly diverse P-loop NTPases fold showed that the typical P-loop architecture is maintained ( Figure 1B). In order to avoid redundancy and utilize the diversity present in the P loop NTPases, we selected a set of 23 NTPase structures at 30% sequence identity cutoff (see methodology). We wanted to look at the impact of sequence diversity on the closeness value of the residues of P-loop and its flanking region. Intriguingly, the highly diverse P-loop NTPases exhibited a similar pattern of high density of conserved high closeness centrality residue positions around Walker A motif, as seen in Ras Super family. Here, the conserved high closeness centrality positions are defined as those positions with statistically significant closeness values (zscore ≥1.0) in at least 60% of the structures of P-loop NTPase fold ( Figure 2 & Table 1). 11 such residue positions around Walker A and its flanking regions showed high closeness value.  (Table 1). Walker A sequence has wider distribution and observed in many proteins that do not bind nucleotides [7]. The structural analysis revealed that these proteins do not form the conspicuous P-loop architecture [7]. To test our prediction, we calculated the closeness value in Walker A sequences that do not form P-loop (Table 2). We did not observe high density of high closeness centrality pattern.
Our results indicate the high density of conserved high closeness residue positions in P-loop and its flanking regions in P-loop fold NTPase and underscore its role in supporting the architecture of P-loop. The study presented is in concord with the observation that highly central residue positions correlate well with active site residues or their neighbors that provide supportive scaffold [13]. However, high closeness value of invariant (G, K, and S/T) residues of Walker A indicates its role in catalysis. P-loop lysine interacts and forms hydrogen-bond with oxygen of γ -phosphate of bound nucleotide and serine/threonine binds with Mg 2+ [16,17]. Recently Grüber et al. [15] demonstrated the role of conserved glycine residues of Walker A motif in guarding the active-site region for nucleotide entrance in archaea-type ATP synthases. The altered conformation of the P-loop resulted in the active-site region being closed to nucleotide entry [15].

Conclusion:
In the context of network, protein structural scaffold and sequence diversity can be visualized as a dramatic change in the type of node, and also the connections between the nodes. Regardless of such diversity, depicted in Ras superfamily and diverse domains of P-loop fold NTPase, the closeness centrality of residue positions in P-loop and its flanking regions are remarkably maintained to be high. Thus, our finding supports the observation that centrality of a residue is maintained evolutionarily to assure the proper functioning of protein [8,13]. We did not find such high centrality residue positions in proteins containing Walker A motif that do not form P-loop. This strengthens the evidence that required geometry of   PDBID  N1  N2  N3  N4  N5  W1  W2  W3  W4  W5  W6  W7  W8  C1  C2  C3  C4  C5   6MHT  _A   PHE  ALA  LYS  THR  GLY  GLY  TYR  LEU  VAL  ASN  GLY  LYS  THR  ARG  LYS  LEU  HIS  PRO  -0.