Sandbox 125

Introduction

Karyopherin Beta 2 (Kapβ2) is an importin that transports various cargo proteins into the nucleus through interactions with nucleoporins, which are proteins of the nuclear pore complex (NPC). One might overlook the significance of this protein but it actually plays a crucial role in the human body by mediating transport of RNA-binding proteins involved in Transcription and RNA Processing, RNA transport and translation. The structure of Kapβ2 is composed of 20 antiparallel helices called HEAT repeats. These HEAT repeats contribute to Kapβ2’s large superhelical shape. The protein is shown to form two arches: one at the N-terminal and the other at the C-terminal. Through recognition of a nuclear localization signal (NLS) located on its cargo, Kapβ2 binds to its cargo via its C-terminal arch. Release of the cargo is mediated by RanGTP, which once bound, leads to a large movement of the 62-residue heat repeat 8 loop into the C-terminal arch. This conformational change results in the dissociation of the cargo as the b2 loop binds the NLS binding site.

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 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 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. 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.3 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 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 either a hydrophobic basic motif or a R-X2-5-P-Y 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 Kapβ2. Interactions of the C-terminal R-X2-5-P-Y motif of the NLS include: Arg284 of the NLS with Glu509 and Asp543 of Kapβ2; Pro288 and Tyr289 of the NLS with Ala380, Ala381, Asp384, Leu419, Ile457, Trp460 and Arg464 of Kapβ2.

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