Kap95p: A Very Busy Protein

BME 220, Protein Bioinformatics, Spring 2009
University of California, Santa Cruz

by Rachel Bingham

Kap95p: An Overview

Figure 1. Space-filling model of 2BKU (PDB_ID) showing the four different chains by color. This figure displays a satellite view from one terminal of the protein to the other, revealing a sweeping spiral in which the importin's cargo would normally fit. This figure shows a complex of Kap95p (its two chains B and D colored blue and cyan, respectively) and the two complexed RanGTP molecules (chains A and C colored dark blue and light blue, respectively.) A cell ships proteins and other cargos into and out of its nucleus at an alarming rate. Over time, this process has become an efficient cycle of catch and release mechanisms aided by a protein ligand known as Ran. Ran binds either GTP or GDP at one time during the course of the cytonuclear import cycle and whichever form of Ran an importin is associated with determines both its configuration and function. Importins, also known as karyopharins, have long been studied and their structures have been fairly well determined mostly by x-ray crystallography. One importin in particular, Kap95p, will be looked at in detail and its structure shown as it was discovered in complex with RanGTP.

The Cycle

Figure 2. Ribbon model of 2BKU (PDB_ID) showing the different structural motifs in the protein. The alpha-helices are observed in pink, beta-sheets in yellow, and others (turns, etc.) in white. This image shows a top-down view of the model looking down the spiral configuration. This model displays the RanGTP(in yellow)-bound conformation that inhibits the binding of a cargo molecule which normally would fit within the spiral-wrap of the protein structure. Import karyopharins floating around in the cytoplasm have one or more specific binding regions available for the binding of RanGDP. RanGDP, in its reduced form, can bind along with a cargo molecule to complete the karyopharin-cargo complex. This particular karyopharin, known as Kap95p, binds both RanGDP and its cargo and then travels through the nucleopore into the nucleus. This process is facilitated by the high negative charge of the karyopharin and the high positive charge of the nucleopore. This electrostatic attraction between importin and nucleopore aids its travel through and into the nucleoplasm. Once inside the nucleus, a RanGEF molecule locates the RanGDP complex bound to Kap95p and activates it, converting the GDP to GTP. This change alters its particular interaction with the importin-cargo complex. RanGDP’s activation causes a conformational change in the importin such that it can no longer hold onto the cargo it is carrying. The cargo then releases from the importin, leaving just the RanGTP bound to the structurally altered importin. As soon as the cargo is gone, the importin together with RanGTP travels back out a nucleopore to be barraged by RanGAP enzymes eager to hydrolyze and deactivate the bound RanGTP. Reduced once more to GDP, RanGDP dissociates from the importin, completing the cycle and leaving the importin free to start the import process with another cargo and RanGDP molecule.

The Players

Figure 3. Ribbon model of 2BKU (PDB_ID) showing a close-up view of the RanGTP complex in light blue(chain C) interacting with the beta-importin subunit colored in cyan (chain D). RanGTP is made up of both beta-sheet and alpha-helix motifs. - Ran is a small protein that has two associated phases: GTP bound and GDP bound. - RanGEF, or guanine nucleotide exchange factor, is a small protein present in high quantities inside the nucleus where it readily converts RanGDP to RanGTP. - RanGAP, or GTPase activating protein, highly populates the cytoplasmic face of nucleoporins where it stands ready to convert any RanGTP it sees to RanGDP. - A nucleoporin is a large cytonuclear transmembrane complex that regulates import, export and diffusion of different molecules (based on size and charge) between the cytosolic and nucleoplasmic regions of the cell.

A Closer Look at the Structure

Figure 4. Ribbon model of 2BKU (PDB_ID) colored as in figure 2 showing a side-ways view of the model. From this angle the importin's spiral can be seen tracing a clockwise configuration going up from the bottom terminus. Kap95p is a beta-importin subunit which binds an alpha-importin subunit, RanGDP, and a cargo molecule. This beta-importin subunit has two unique chains and two interacting RanGTP subunits. A remarkable symmetry of the molecule is displayed in figure 5. In the simplest of words, Kap95p forms a spiral staircase as shown in figure 4 around its cargo protein, wrapping around to carry it through the nuclearpore. An importin’s function is largely due to the intermolecular interactions between the many alpha-helices making up the main structural motif in this protein. Its precise mode of transporting cargo through the nucleopore remains a topic of debate. However one currently circulating theory proposes a mechanism of jumping from one docking site along the walls of the nucleopore to another docking site. According to a random stochastic modeling that accompanies this theory, the docking site the importin jumps to may either be closer to or further from its intended exit goal. Phenylalanine-glycine (F-G) repeat motifs along the nucleoporin wall are what the importin recognizes as potential docking sites. The phenylalanine residues found within these motifs nestle their side chains in pockets on the surfaces of the importin's structure. Figure 5. Ribbon model of 2BKU (PDB_ID) colored as in figure 2 showing the vertical symmetry of the model. Symmetry of this model revolves around a vertical axis where the right half of the protein could fold along the central vertical axis and superimport (in terms of backbone positioning) itself onto the left half of the protein. Figure 6. Ribbon model of 2BKU (PDB_ID), colored by chain as in figure 3, showing an overall view of the protein and how RanGTP plays an important role in its structural conformation.

Some Notes on RasMol:

RasMol was the tool of choice for creating the images above. The color feature was particularly useful for showing the mass of alpha-helices in the model and where RanGTP binds because of its unique beta-sheets. One thing that was frustrating was the inability to obtain a static picture that easily displays the protein’s 3-D spiral shape. The quality of the images RasMol provides is sufficient enough. However, if PyMol had a working version of its visualizer that was compatible with Windows Vista that likely would have been my first choice.

References

Roger Sayle and E. James Milner-White. "RasMol: Biomolecular graphics for all", Trends in Biochemical Sciences (TIBS), September 1995, Vol. 20, No. 9, p. 374.
Lee, S. (2005). Structural basis for nuclear import complex dissociation by RanGTP. Nature, 435(7042), 693-696.

Last Updated: 03 May 2009