PDBsum entry 5c46

Transferase/signaling protein

PDB id: 5c46

Contents

Protein chains

469 a.a.

175 a.a.

Ligands

SO4 ×2

GSP

Metals

_MG

Waters ×4

PDB id:

5c46

Name:

Transferase/signaling protein

Title:

Crystal structure of an engineered construct of phosphatidylinositol 4 kinase iii beta in complex with gtp gamma s loaded rab11

Structure:

Phosphatidylinositol 4-kinase beta. Chain: e. Synonym: ptdins 4-kinase beta,npik. Engineered: yes. Mutation: yes. Ras-related protein rab-11a. Chain: f. Synonym: rab-11,yl8. Engineered: yes.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: pi4kb, pik4cb. Expressed in: escherichia coli. Expression_system_taxid: 469008. Gene: rab11a, rab11.

Resolution:

2.65Å R-factor: 0.218 R-free: 0.246

Authors:

J.E.Burke,M.L.Fowler

Key ref:

M.L.Fowler et al. (2016). Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11. Protein Sci, 25, 826-839. PubMed id: 26756197 DOI: 10.1002/pro.2879

Date:

17-Jun-15 Release date: 20-Jan-16

Protein chain

Q9UBF8  (PI4KB_HUMAN) -  Phosphatidylinositol 4-kinase beta from Homo sapiens

Seq: 816 a.a.

Struc: 469 a.a.

Protein chain

P62491  (RB11A_HUMAN) -  Ras-related protein Rab-11A from Homo sapiens

Seq: 216 a.a.

Struc: 175 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: Chain E: E.C.2.7.1.67 - 1-phosphatidylinositol 4-kinase.
• Pathway: 1-Phosphatidyl-myo-inositol Metabolism
• Reaction: a 1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol) + ATP = a 1,2-diacyl- sn-glycero-3-phospho-(1D-myo-inositol 4-phosphate) + ADP + H⁺

1,2-diacyl-sn-glycero-3-phospho-(1D-myo-inositol) + ATP (Bound ligand (Het Group name = GSP) matches with 90.91% similarity) = 1,2-diacyl- sn-glycero-3-phospho-(1D-myo-inositol 4-phosphate) + ADP + H(+)

• Enzyme class 3: Chain F: E.C.3.6.5.2 - small monomeric GTPase.
• Reaction: GTP + H2O = GDP + phosphate + H⁺

GTP (Bound ligand (Het Group name = GSP) matches with 93.94% similarity) + H2O = GDP + phosphate + H(+)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1002/pro.2879

Protein Sci 25:826-839 (2016)

PubMed id: 26756197

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

M.L.Fowler, J.A.McPhail, M.L.Jenkins, G.R.Masson, F.U.Rutaganira, K.M.Shokat, R.L.Williams, J.E.Burke.

ABSTRACT

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches.