Structure and Backbone Dynamics of Vanadate-Bound PRL-3: Comparison of ¹⁵N Nuclear Magnetic Resonance Relaxation Profiles of Free and Vanadate-Bound PRL-3

Abstract

Phosphatases of regenerating liver (PRLs) constitute a novel class of small, prenylated phosphatases with oncogenic activity. PRL-3 is particularly important in cancer metastasis and represents a potential therapeutic target. The flexibility of the WPD loop as well as the P-loop of protein tyrosine phosphatases is closely related to their catalytic activity. Using nuclear magnetic resonance spectroscopy, we studied the structure of vanadate-bound PRL-3, which was generated by addition of sodium orthovanadate to PRL-3. The WPD loop of free PRL-3 extended outside of the active site, forming an open conformation, whereas that of vanadate-bound PRL-3 was directed into the active site by a large movement, resulting in a closed conformation. We suggest that vanadate binding induced structural changes in the WPD loop, P-loop, helices α4-α6, and the polybasic region. Compared to free PRL-3, vanadate-bound PRL-3 has a longer α4 helix, where the catalytic R110 residue coordinates with vanadate in the active site. In addition, the hydrophobic cavity formed by helices α4-α6 with a depth of 14-15 Å can accommodate a farnesyl chain at the truncated prenylation motif of PRL-3, i.e., from R169 to M173. Conformational exchange data suggested that the WPD loop moves between open and closed conformations with a closing rate constant k(close) of 7 s(-1). This intrinsic loop flexibility of PRL-3 may be related to their catalytic rate and may play a role in substrate recognition.

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

“…4, B and C, whereas Ec-ACP has only two phenylalanines, Phe 28 and Phe 50 . As commonly shown in most ACP structures, Val 6 , Phe 53 , and Ile 75 at the top end of the hydrophobic cavity of ACP have close hydrophobic contacts with each other (Fig. 4B).…”

“…In the final structural calculations, a total of 100 structures for ACP were calculated using the program CYANA, and 20 structures with the lowest target functions were selected and superimposed (Fig. 4A) 53 , which play an important role in hydrophobic packing of the helical bundle, as shown in Fig. 4, B and C, whereas Ec-ACP has only two phenylalanines, Phe 28 and Phe 50 .…”

“…The recycle delay and proton saturation times in the hNOE measurement were 4.5 and 3.0 s, respectively. All relaxation spectra were acquired and analyzed as described in our previous reports (53,54). hNOE was calculated from the ratio of peak heights with and without proton saturation pulses.…”

Structural Characterization of an ACP from Thermotoga maritima: Insights into Hyperthermal Adaptation

“…For side chain assignment, CC(CO)NH, HBHA(CO)NH, H(CCO)NH, and HCCH-TOCSY spectra were obtained. The assignments were confirmed by 1 H-15 N-1 H and 1 H-13 C-1 H NOESY-HSQC spectra [18,[59][60][61][62]. All NMR spectra were processed with NMRPipe [63] and analyzed with NMRFAM-Sparky [64].…”

Targeting phosphatases of regenerating liver (PRLs) in cancer

“…3B ), and PAC-1 adopts an intermediate conformation between open and closed. A study comparing free and vanadate-bound PRL-3 structures using NMR ( K. W. Jeong et al, 2014 ) showed that PRL-3 was able to adopt a closed conformation upon vanadate addition, with Asp 72 , Cys 104 and Arg 110 close together. PRL-3 therefore likely undergoes substantial conformational rearrangement upon substrate binding, which pulls Asp 72 near to catalytic Cys 104 .…”