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. 2017 Jun 20;112(12):2529-2539.
doi: 10.1016/j.bpj.2017.03.042.

Prp40 Homolog A Is a Novel Centrin Target

Adalberto Díaz Casas  1 , Walter J Chazin  2 , Belinda Pastrana-Ríos  3
Affiliations

Prp40 Homolog A Is a Novel Centrin Target

Adalberto Díaz Casas et al. Biophys J. .

Abstract

Pre-mRNA processing protein 40 (Prp40) is a nuclear protein that has a role in pre-mRNA splicing. Prp40 possesses two leucine-rich nuclear export signals, but little is known about the function of Prp40 in the export process. Another protein that has a role in protein export is centrin, a member of the EF-hand superfamily of Ca2+-binding proteins. Prp40 was found to be a centrin target by yeast-two-hybrid screening using both Homo sapiens centrin 2 (Hscen2) and Chlamydomonas reinhardtii centrin (Crcen). We identified a centrin-binding site within H. sapiens Prp40 homolog A (HsPrp40A), which contains a hydrophobic triad W1L4L8 that is known to be important in the interaction with centrin. This centrin-binding site is highly conserved within the first nuclear export signal consensus sequence identified in Saccharomyces cerevisiae Prp40. Here, we examine the interaction of HsPrp40A peptide (HsPrp40Ap) with both Hscen2 and Crcen by isothermal titration calorimetry. We employed the thermodynamic parameterization to estimate the polar and apolar surface area of the interface. In addition, we have defined the molecular mechanism of thermally induced unfolding and dissociation of the Crcen-HsPrp40Ap complex using two-dimensional infrared correlation spectroscopy. These complementary techniques showed for the first time, to our knowledge, that HsPrp40Ap interacts with centrin in vitro, supporting a coupled functional role for these proteins in pre-mRNA splicing.

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Figures

Figure 1
Figure 1
Amino acid sequence analysis of HsPrp40A. (A) Sequence alignment of CBSs of known centrin targets including HsXPC (UniProt: Q01831), HsSfi1 (UniProt: A8K8P3), HsPOC5 (UniProt: Q8NA72), ScSac3 (UniProt: P46674), and the novel target HsPrp40A (UniProt: O75400). HsSfi1, HsPOC5, and ScSac3 are in a reversed orientation (C- to N-terminal end) to highlight the conserved hydrophobic residues. (B) Helical wheel of the CBS within HsPrp40A comprising the amino acid sequence beginning at Lys524 through Met541. The hydrophobic triad W1L4L8 is identified in gray. The helical wheel representation was generated using an online resource at http://kael.net/helical.htm. (C) Sequence alignment of Prp40A in H. sapiens, M. musculus (UniProt: Q9R1C7), D. renio (UniProt: Q7ZUE4), X. laevis (UniProt: Q08AZ7), and C. reinhardtii (available at the Orthologous Matrix database under accession number CHLRE00747), including ScPrp40 (UniProt: P33203).
Figure 2
Figure 2
Binding isotherm of the titration of HsPrp40Ap by Hscen2 at 25°C. (A) Power data and (B) integrated enthalpy are fitted to a one-binding-site model.
Figure 3
Figure 3
Relative affinities of the interaction between Crcen with HsPrp40Ap at (A) 20°C, (B) 25°C, (C) 30°C, and (D) 35°C, showing the power data (upper) and the integrated enthalpy (lower) fitted to a one-binding site model.
Figure 4
Figure 4
2D IR correlation spectroscopy of the 13C-Crcen-HsPrp40Ap complex (1:1 molar ratio). (A) Overlaid FT-IR spectra and (B) synchronous and (C) asynchronous plots within the spectral region 1700–1500 cm−1 and over the 5–90°C temperature range with 5°C temperature intervals. In (A), the black and red lines correspond to 5°C and 90°C, respectively.
Figure 5
Figure 5
Sequential order of molecular events during the thermal perturbation of the 13C-Crcen-HsPrp40Ap complex (1:1 molar ratio) over the temperature range 5–90°C. The key to complex formation is the inter-molecular salt bridge interactions between HsPrp40Ap’s negatively charged residues and centrin’s Arg residues, as well as the requirement of the hydrophobic interaction within the binding interface. The text in bold corresponds to HsPrp40Ap’s vibrational modes.
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