Structural, biochemical and computational studies of KIBRA protein-protein and protein-phospholipid interactions that are important for memory

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry

Abstract

KIBRA and the molecular mechanism of memory
Our memories define who we are. Understanding the mechanisms behind the acquisition, storage and recall of memories is consequently a neuroscience holy grail. Memory depends on the interplay of different types of proteins (e.g. receptors, channels, enzymes, scaffold proteins). We will study KIBRA, a scaffold protein that is important for memory and that is linked to Alzheimer's disease. Since many details of how KIBRA functions in neurons (brain cells) are lacking, we will use experimental and computational methods to study KIBRA interactions with partner proteins and membranes (permeable biological boundaries between different parts of a cell or between cells).

PKMzeta is crucial for long term memory storage
The mechanisms of short and long term memory differ. The pivotal player in long term memory storage is a protein called PKMzeta which works by modifying other proteins. Modification by PKMzeta of receptor proteins, especially AMPA receptors, at the surface of neurons, for example, causes the receptors to move to the postsynaptic membrane where they contribute to electrical or chemical signalling between neurons to maintain memories. PKMzeta acts like a conveyor belt to carry AMPA receptors to the synapse. Modulation of PKMzeta activity can disrupt or enhance memory.

KIBRA-PKMzeta interaction is vital for PKMzeta function
Our collaborators have shown that KIBRA interaction with PKMzeta is crucial for PKMzeta's conveyor belt action. We will define details of KIBRA-PKMzeta interaction, e.g. the nature of the interface between the proteins, role of protein movement, and which protein components are most important for the interaction.

Other KIBRA interactions that are important for memory
We will study two other KIBRA interactions: with the key neuronal proteins Dendrin and Synaptopodin that are organisers of the molecular skeleton that gives neurons their characteristic shape; and KIBRA's interaction with membranes that maintains KIBRA in the correct location within neurons. Different parts of KIBRA are involved in its interactions with PKMzeta, Dendrin and Synaptopodin, and membranes.

Methods
We will use the complementary characteristics of multiple methods. NMR exploits the magnetic properties of nuclei to provide information about the shapes, shape changes and interactions of proteins at many locations throughout proteins and across a wide range of timescales (picoseconds to minutes). X-ray crystallography provides higher resolution shape information but less insight into dynamic behaviour. Other methods tell us about the strength and dynamics of protein interactions with other molecules. Computer simulations of protein behaviour provide insights that are not available from experiment alone, aid interpretation of experimental data and inform design of new experiments. Combining our study of molecules with our collaborator Joachim Kremerskothen's parallel experiments on cells and animals should lead to deep insight into KIBRA function.

Long term goal: treatments for addictions, phobias, stress and anxiety disorders, and memory decline
We will use our results to guide alterations in KIBRA that disrupt/enhance its interactions. Dr Kremerskothen will study how these KIBRA alterations affect neuron functions and memory processes in animals. This could help in the development of new molecules that affect memory. One existing molecule called ZIP that inhibits PKMzeta, for example, erases all long term memories yet permits formation of new memories. Improved understanding of memory-related molecular interactions such as those of KIBRA could help to develop molecules that specifically disrupt individual long term memories rather than all long term memories, for example to treat addiction, phobias, stress and anxiety disorders, or develop molecules that enhance memory performance in the elderly, Alzheimer's patients, or physical trauma victims.

Technical Summary

We will advance understanding of the molecular basis of KIBRA's role in memory as follows:

Details underlying C2 domain role in KIBRA membrane trafficking
For normal and variant KIBRA C2 domains, we will use NMR, isothermal titration calorimetry (ITC) and X-ray crystallography (XRC) to measure Ca2+ binding properties (affinity, stoichiometry, cooperativity, occupation order of Ca2+ sites (up to 3), and Ca2+-induced structural changes). Phospholipid specificity will be probed by NMR and ITC/surface plasmon resonance (SPR) measurement of normal and variant C2 binding to monodisperse analogues of phosphatidyl-inositol phosphates, -serine and -choline. We will use multiscale simulations to study membrane penetration depth, membrane-binding geometry, lipid determinants and role of different forces in normal and variant C2-membrane interaction.

KIBRA WW domains
We will determine whether the 2 tandem WW domains of KIBRA are cooperative or independent, ascertain the role of the atypical (Ile instead of Trp) second WW and establish the selectivity and structures of single and tandem WW interactions with single and multiple PPxY motif ligands from Dendrin and Synaptopodin, postsynaptic cytoskeleton organisers associated with neuronal synaptic plasticity.

KIBRA-PKMzeta association
PKMzeta is pivotal for long term memory storage. KIBRA-PKMzeta association is vital for PKMzeta function. We will use ITC, thermal shift, enzyme assays, XRC, NMR, and mutagenesis to delineate thermodynamics, kinetics, structural details and key residues of KIBRA-PKMzeta interaction. Starting from the known PKMzeta binding fragment, KIBRA residues 953-996, we will find the minimal binding site. Our data will guide design of peptides that bind PKMzeta that JK will inject into rat brain and test for memory and behaviour effects.

Our studies will synergise with JK's parallel cell and in vivo studies towards detailed understanding and modulation of KIBRA function in memory.

Planned Impact

The research fits with BBSRC's Bioscience Underpinning Health priority and BBSRC Grand Challenge 3 - Fundamental bioscience enhancing lives and improving wellbeing (BBSRC Delivery Plan 2011-15). The research has benefit and interest in several areas, particularly due to its potential to contribute to ongoing research to find new treatments for memory-related afflictions such as addiction, phobias, stress and anxiety disorders, and memory decline.

Economic and social burden of addictions (e.g. smoking, alcohol, illegal drugs, gambling)
Addictions exert a huge socio-economic burden. In 2005, for example, 109000 UK deaths were attributable to smoking with £5.2bn NHS costs. ("The burden of smoking-related ill health in the UK", Tobacco Control 18 (2009) 262). Global alcohol-related costs were estimated at $210-665 billion in 2002. ("The global economic burden of alcohol", Drug Alcohol Rev. 25 (2006) 537). Research reported in The Independent (Feb 2002) showed that "drug addicts cost the NHS, the state benefits system and the criminal justice system around £6.8bn a year. The social costs of drug addiction, mainly the cost to victims of crime, amount to a further £12bn annually."

Economic and social burden of stress and anxiety disorders, phobias and memory loss
PTSD affects 2.6-10% of British soldiers deployed to Afghanistan and Iraq (669-2548 soldiers per year; Select Committee on Defence) and up to 30% of road accident victims. In the USA, anxiety disorders cost more than $42 billion annually ("The economic burden of anxiety disorders," J. Clin. Psych. 60, 1999) and the National Institute of Mental Health reported in 2005 that 8.7-18.1% of Americans suffer from phobias. Memory loss due to age, disease and trauma exerts a huge burden; e.g. the Dementia 2010 report (dementia2010.org) was headlined "Dementia costs UK plc £23 billion a year". This cost will rise as the proportion of UK society living beyond 65 increases rapidly.

Pharmaceutical and biotech sector
e is ongoing academic and industry research into memory modulator compounds e.g. propranolol (Nat. Neurosci. 12 (2009) 256). Zeta inhibitory peptide (ZIP; 13 residues) that inhibits PKMzeta can permanently erase all long term memories without preventing new memory formation (Science 317 (2007) 951). PKMzeta overexpression, moreover, enhances memory (Science 331 (2011) 1207). In 10-20 years, detailed understanding of memory-related molecular interactions such as those of KIBRA could be used by pharmaceutical/biotech/spin-out companies to develop biological or chemical molecules that specifically disrupt individual memories, for example to treat addiction, stress and anxiety disorders, or phobias, and molecules that enhance memory to treat memory decline due to ageing, disease or trauma.

Public sector and general public
The quality of life of many people would be improved by new drugs to treat memory-related afflictions such as addiction, stress and anxiety disorders, phobias and memory decline. Such treatments could, for example, significantly reduce NHS costs, reduce addiction-associated crime and other social problems, help military PTSD sufferers, and allow more people to enjoy a healthy old age. In the much shorter term, local schools and interested public will benefit from the proposed outreach activities (Pathways to Impact).

People: lab and transferable skills
The PDRA and PhD/undergrad students will benefit from substantial training from PIs and collaborators who have diverse expertise and interests, and from access to state-of-the-art equipment, for example national NMR facilities, local X-ray facilities, Diamond Synchrotron, and Oxford Protein Production Facility. The PDRA and students will also acquire transferable skills both from working on the project and through attending courses e.g. project management (including budgetary aspects), written and oral communication skills including public and media engagement, and enterprise.
 
Description KIdney- and BRAin-expressed protein (KIBRA, also called WWC1 for WW-and-C2-domain-containing-protein-1) is a large, multi-functional scaffold protein with about twenty reported binding partners. Numerous studies have demonstrated links between KIBRA and memory, cognition and neurological disorders in humans and in rodent models. A single nucleotide polymorphism (SNP), rs17070145, within the ninth intron of KIBRA, for example, has been implicated in human cognition.

KIBRA has two N-terminal WW domains, a C2 domain, one or more putative coiled coil regions, a glutamic acid-rich motif, an atypical PKC (aPKC) binding region and a C-terminal PDZ binding motif. A recently identified variant that arises from two further SNPs (rs3822660G/T and rs3822659T/G) in human KIBRA results in substitution of two adjacent residues (M734I, S735A) in the C2 domain. These exonic SNPs affect cognitive performance and there is almost complete linkage disequilibrium between them and the previously mentioned intronic SNP rs17070145, meaning that the alleles are non-randomly associated. Our studies of KIBRA C2 domain have included 4 forms of the C2 domain, comprising wild type and C771A mutant forms of the "normal" and "variant" C2 domains that occur in the human population "wild type", M734I/S735A variant, and C771A mutants of both.

Our main findings to date include:
1. X-ray crystallography structure of KIBRA C2 domain as a calcium-free, anti-parallel dimer.
The extant crystal structure of human KIBRA C2 (PDB code 2Z0U) is a calcium-free parallel dimer with monomers linked via an inter-domain disulphide bond involving C771. Our analysis, however, indicates that the inter-chain contact region in the parallel dimer is strikingly lacking in good non-covalent interactions. Also, we doubt that such an inter-molecular disulphide bond will persist in the reducing environment of the cytoplasm. We crystallised the C771A mutant C2 domain. This forms a calcium-free anti-parallel dimer in the crystal that displays a sensible network of inter-chain polar interactions, so it looks more like a proper dimer interface than the V-shaped parallel dimer seen in PDB entry 2Z0U. We also note previous evidence for anti-parallel dimerisation of KIBRA via the C2 domain (Neuroscience 155 (2008) 1165). The lack of bound calcium, even when crystals were soaked with calcium chloride solution, is potentially significant as C2 domains are most often calcium-dependent membrane association domains.

2. Weak calcium binding by KIBRA C2 domain. NMR titrations and X-ray crystallography (please see 1. above) indicate that KIBRA C2 domain has low calcium affinity relative to many other C2 domains.

3. Unusual mode of phosphoinositide binding by KIBRA C2 domain. Our ongoing analysis of data from NMR titrations and X-ray crystallography indicates that KIBRA C2 domain interacts with phosphoinositides in an unusual, perhaps unique, manner for a C2 domain. We note further that both NMR titrations and X-ray crystallography indicate that C2-phosphoinositide interaction is calcium-independent.

4. The wild type and variant C2 domains are structurally very similar but do show some different characteristics. NMR samples of the wild type C2 domain, for example, show evidence of aggregation more rapidly than those of variant C2 domain at similar concentrations. In analytical ultracentrifugation, furthermore, wild type C2 appeared as an unresolved mixture of species whereas variant C2 appeared as two well defined species with molecular weights consistent with a monomer and a dimer. X-ray crystal structures indicate that the phosphoinositide binding sites are the same for wild type and variant C2 domains.

5. Membrane binding mechanism
Antreas Kalli in Mark Sansom's group at Oxford has been simulating structures of the KIBRA C2 domains. In line with our data, he found that the anti-parallel dimer is very stable in solution whereas the parallel dimer is more dynamic. KIBRA C2 has a large positive surface on the opposite end of the domain to the Ca2+ binding loops. In membrane binding simulations this interacts strongly with the anionic membrane, placing the Ca2+ binding loops away from the membrane (net charge of the membrane is -201). In contrast, in the Ca2+ binding loop region there is a negative surface and apparently the presence of Ca2+ ions forms a bridge between this anionic region and the anionic membrane and thus in the simulations with two bound Ca2+ ions there are two populations: one with the Ca2+ binding loop region in contact with the membrane and one with the positive region on the opposite end of the domain in contact with the membrane. The latter is an unusual membrane binding orientation for a C2 domain and seems to strengthen the idea that KIBRA C2 domain exhibits some unusual properties.

We are currently integrating these and other simulation data with our experimental data to construct one or two manuscripts on KIBRA C2 domain.
Exploitation Route Our groundwork on PKMzeta production will be very useful going forward as this enzyme is a central molecular engine of long term memory (Science 2006;313:1141; Science 2007;317:951; Science 2011;331:1207). Potential lines of research to improve molecular level understanding of the structure, regulation and modulation of this key enzyme for long term memory that could be taken forward as a result of our work on production of two PKMzeta constructs include:
1. Professor Todd Sacktor's group (SUNY, USA) observes clear dominant negative interaction between inactive PKMzeta and active (phosphorylated) PKMzeta, both in cells and in vitro (pers. comm.). This kinase dimerisation might be very important for long term memory, in line with Crick's hypothesis on the molecular basis of long term memory (Crick F. Nature 1984;312:101). It will therefore be important to establish whether and how PKMzeta dimerises.
2. We also have seven PKMzeta mutants (phosphorylation site and dominant negative mutants e.g. Science 2011;331:1207) that we have not had time to characterise. Molecular and cellular studies of these mutants will be important to understand the role of phosphorylation and the K281W dominant negative mutation.
3. KIBRA binds, co-localises with and is phosphorylated by PKMzeta, and direct interaction between a twenty amino acid KIBRA motif (residues 956-975) and PKMzeta counteracts proteasomal degradation of PKMzeta. Also, KIBRA plays a crucial role in PKMz?t??control of neuroreceptor trafficking. Analysis of KIBRA-PKMzeta interaction was part of the grant proposal.
4. Dr Mildred Acevedo-Duncan, University of South Florida, is developing a new, PKMzeta-specific inhibitor. Dr Acevedo-Duncan's group has previously developed an inhibitor specific to PKCiota (Int J Biochem Cell Biol. 2011;43:784) (PKMzeta and PKCiota share 74% sequence identity). Study of PKMzeta interaction with this novel specific inhibitor will be an extremely powerful tool in memory studies, for example for molecular memory modulator design.

In addition, our work on structure and function of KIBRA C2 domain could be continued to understand the molecular mechanism by which particular KIBRA alleles affect memory and cognition. This could include experimental investigation of KIBRA C2-membrane interaction using nanodiscs and NMR spectroscopy to complement our KIBRA C2-membrane simulations. Experiments started by our collaborator, Joachim Kremerskothen (JK), could also be completed using our C2 domain data. JK showed, for example, that intracellular, vesicle-associated transport of GFP-KIBRA depends on a functional C2 domain, and C2 domain ablation results in increased average speed of transport. Vesicle tracking in transfected cells could be used to determine the effect of mutating residues known from our studies to be important for phosphoinositide binding such as L666, I677 and L678. Such C2 domain mutants could similarly be investigated for their effect on directional migration of transfected cells in a wound healing assay (JK showed that KIBRA C2 domain overexpression negatively affects directional migration of transfected cells).
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Analysis of KIBRA C2 domain interaction with membranes; Mark Sansom lab 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental data on KIBRA C2 interaction with calcium and phosphoinositides
Collaborator Contribution Simulation of KIBRA C2 dimerisation, interaction with calcium, and interaction with membranes
Impact No outputs yet but manuscript is in preparation. Combines experimental with computational biochemistry.
Start Year 2013
 
Description OPPF 
Organisation Research Complex at Harwell
Department Oxford Protein Production Facility-UK (OPPF-UK)
Country United Kingdom 
Sector Academic/University 
PI Contribution Design of PKMzeta protein expression constructs.
Collaborator Contribution Expression of PKMzeta both alone and co-expressed with PDK1 in insect and mammalian cells. Crystallisation screens.
Impact No outputs/outcomes yet.
Start Year 2012
 
Description Rieko Ishima 
Organisation University of Pittsburgh
Department School of Medicine Pittsburgh
Country United States 
Sector Academic/University 
PI Contribution Provision of purified KIBRA C2 domains (wild type and variant).
Collaborator Contribution Acquisition and analysis of NMR relaxation data for investigation of C2 domain dynamics.
Impact Review article co-authored by Ishima and Bagby, in press: Protein dynamics revealed by CPMG dispersion, to be published in Modern Magnetic Resonance (Springer)
Start Year 2012
 
Description Todd Sacktor lab 
Organisation State University of New York
Country United States 
Sector Academic/University 
PI Contribution Expression and purification of wild type PKMzeta for structure-function analysis e.g. interaction with KIBRA-derived peptides. Crystallisation screens (not yet successful). Insertion of seven PKMzeta mutants into protein expression vectors.
Collaborator Contribution Intellectual input. Provision of mutant cDNAs.
Impact No outputs/outcomes yet.
Start Year 2013
 
Description School visit (Bristol) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation, discussion and activities related to memory and cognition to sixth form at Chipping Sodbury School. Increased interest in science as a career option.
Year(s) Of Engagement Activity 2015