Sandbox 125

Identification of the Cargo and Transport Through the NPC

In eukaryotes, proteins must be transported in and out of the nucleus. This nucleocytoplasmic transport of proteins across the nuclear envelope must occur through the gateway of the NPC. The NPC is a large structure consisting of 456 constituent binding proteins called nucleoporins (Nups).1 Movement through the NPC is facilitated transport that relies on interaction with specific Nups. Importins and exportins are proteins that aid this facilitated transport by both binding to a specific cargo to be transported and interacting with specific Nups located in the central channel of the NPC.2

Karyopherin Beta 2 (KapB2) is a group of proteins that is composed of both importins and exportins. Importins are proteins that carry cargos into the nucleus while exportins serve the opposite function. As of today, twenty different Kapβs have been identified. Each of these Kapβs is capable of recognizing and transporting a specific group of cargos. In order to bind to its cargo a Kapβ has to recognize a Nuclear Localizaton or Export Signal (NLS or NES) is located in the polypeptide chain of the cargo. These signals can vary from 7 amino acids to longer than 100 amino acids in length.

An importin, such as Kapβ2, binds to a specific cargo by recognition of an NLS and carries the cargo through the NPC by interacting with intrinsically disordered Nucleoporins called FG-Nups. FG-Nups line the passageway of the NPC and contain repeats of phenylalanine and glycine. These unstructured FG-Nups form a low-density cloud within the central channel extending from the cytoplasm to the nucleoplasm. The cloud acts as an effective exclusion filter for those particles that do not contain FG repeat binding sites. This is referred to as the zone of selectivity.

Structure of Kapβ2

Kapβ2 is a superhelix comprised of 20 HEAT repeats (the name HEAT derives from Huntington, Elongation factor 3 A subunit of protein phosphatase 2A and Tor1 kinase), each of which consists of two anti-parallel helices (shown in red). The electrostatic potential of the internal surface of Kapβ2 superhelix at the C-terminal arch is negative. HEAT repeats 9-13 and 14-18 form the binding site of Kapβ2 cargos while repeats 1-8 constitute the Ran GTPase binding site. Ran GTPase, a small 216-residue protein, is found more frequently in the nucleus and enables cargos to be released from Kapβ2.

Kapβ2 Binding and Conformational Change

The NLSs located on KapB2 cargos are named the PY-NLS (Proline/Tyrosine-Nuclear Localization Signal) and they bind to the C-terminal arch of KapB2.

Recognition of the PY-NLS by Kapβ2 follows certain guidelines:

(i) PY-NLS, when not bound to Kapβ2, lacks a secondary structure.

(ii) PY-NLS has an overall positive charge allowing for electrostatic compatibility with Kapβ2.

(iii) General sequence for the PY-NLS is a hydrophobic basic motif and a RX2-5PY motif at the C-terminus.

The PY-NLS of 2H4M contains a hydrophobic rather than a basic N-terminal motif. Hydrophobic interactions at the N-terminal motif of the PY-NLS include:

Phe273, Gly274, Pro275 and Met276 of the NLS with Trp730 and Ile773 of KapB2. Interactions of the C-terminal RX2-5PY motif of the NLS include: Arg284 of the NLS with Glu509 and Asp543 of KapB2.; Pro288 and Tyr289 of the NLS with Ala380, Ala381, Asp384, Leu419, Ile457, Trp460 and Arg464 of KapB2.

Upon binding Kapβ2,the NLS gains structure, conforms to and makes contact with the internal surface of the KapB2 C-terminal arch.

How does Kapβ2 pass through the NPC?

Once the cargo binds to Kapβ2, the complex travels through the NPC by interactions with the FG-Nups.

How does Kapβ2 release its cargo?

Ran is a GTP binding protein that is found in greater concentrations in the nucleus than in the cytoplasm. In the nucleus Ran Guanine Nucleotide Exchange Factor (ranGEF) is a protein that catalyzes the dissociation of GDP from Ran and subsequent phosphorylation. Ran-GTP undergoes conformational changes that allow it to have a greater affinity for Kapβ2 than Ran-GDP. These conformational changes occur in Ran between residues 30-47 and residues 65-80.

RanGTP has an overall positive charge allowing it to bind to the highly negative core of the N-terminal arch Kapβ2. There are two distinct regions of polar interaction between KapB2 and RanGTP. These regions of Kapβ2 are defined as the N Interface found within HR1-3, and the Central Interface found within HR6, HR7, 62-residue loop of HR8, HR13 and HR14.

Polar interactions at the N Interface include Ser22, Arg31, Glu161, Asp 164 and Ser165 of KapB2, and Trp64, Val45, Arg110, and Arg106 of RanGTP.

Polar interactions at the Central Interface include Asn271, Glu275, Glu278, Glu332, Glu333, Asp334, Arg336, His340, Glu363, Ile364, Asp366, Asp367, Ile370, Ser371, Trp373, Lys377, Asp639, Glu682 of KapB2 and Asn143, Lys141, Arg140, Asn154, Asn156, Lys159, Asp148, Tyr155, Lys127, Lys134, Lys132, Arg129, His139, Tyr 146, Gln145, Asn143, Arg140, and Lys127 of RanGTP.

When RanGTP binds to Kapβ2 conformational changes occur within the N-terminal arch and within a long (62 residue) loop of HEAT repeat 8. It is the changes in the 62-residue loop (residues 311-373) that is important in the dissociation of the NLS. It is thought that the 62- residue loop binds into the NLS binding site thus releasing the NLS.