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Protein-Protein InteractionsIntroduction

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Experimental Methods

Interaction Experiments: Examples

• Native (chicken) complex purified from heart mitochondria

• Structure determined by X-ray crystallography to 3.0A

• 9 (out of 11) subunits identified/localized, at least partially, within the structure

Zhang et al. Nature 392:677-684(1998); PMID: 9565029

Electron density

PDB: 1BCC

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Interaction Experiments: Examples

• Fusion of a fragment of human 2-AR with lysozyme fusion expressed in insect (Sf9) cells

• Gs (bovine), G1 (rat, His-tagged) and G2

(bovine) co-expressed in insect (HiFive) cells • Camel nanobody expressed in E. coli • In vitro complex reconstitution• Structure determined by X-ray crystallography

to 3.2A

Rassmussen et al. Nature 477:549-555(2011); PMID: 21772288

PDB: 3SN6

PDB: 3SN6

Interaction Experiments: Examples

• Native expression (spinach)• Cryo-EM structure determination

Hahn et al. Science 360:eaat4318 (2018); PMID: 29748256

PDB: 6FKF

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Interaction Experiments: Examples

• Conformational states

Hahn et al. Science 360:eaat4318 (2018); PMID: 29748256

PDB: 6FKF PDB: 6FKH PDB: 6FKI

Interaction Experiments: Examples

Alfieri et al. Nature 536:431-436 (2016); PMID: 27509861

• Heterologous expression (human proteins/insect cell expression)

• In vitro complex reconstitution• Cryo-EM structure determination• Alternative conformational states

PDB: 5LCW

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Interaction Experiments: Examples

• Heterologous protein expression (insect cells)• In vitro complex reconstitution• Size exclusion chromatography of the final purified

complex

Alfieri et al. Nature 536:431-436 (2016); PMID: 27509861

WikipediaSize Exclusion Chromatography

Interaction Experiments: Examples

• Overexpression (human) of doubly-tagged (FLAG & HA) human H2A, H2A.Z & ANP32E proteins

• Tandem affinity purification of interacting partners • Mass spectrometric and/or Western blot protein

identification

Affinity ChromatographyObri et al. Nature 505:648-53 (2014); PMID: 24463511

MS

Western

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Interaction Experiments: Methods

• Purified fragment (1-149; His-tagged) of rat CNGA3 expressed in E. coli

• Purified rat CDH23 (+/- exon68; His-tagged) expressed in E. coli

Surface Plasmon Resonance

Selvakumar et al. JBC 288:7215-7229 (2014); PMID: 23329832

Bind Wash

Immobilized CNGA3

CDH23 (+68)

CDH23 (-68)

buffer

CDH23 (+68)

Interaction Experiments: Examples

• Heterologous (E. coli) expression of Salmonella (SopE2) and human (Cdc42) protein fragments

• In vitro complex formation by purified proteins• Monitor perturbation of SopE2 NMR spectra by Cdc42 protein

Interaction-Induced Property Change

Williams et al. Biochem. 43:11990-1008 (2004); PMID: 15379540

NMR

X-ray

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B

Interaction Experiments: Examples

• Overexpression (human) of doubly-tagged (FLAG & HA) human H2A, H2A.Z & ANP32E proteins

• Tandem affinity purification of interacting partners • Mass spectrometric and/or Western blot protein

identification

In vivo Complementation Assays

Selvakumar et al. JBC 288:7215-7229 (2014); PMID: 23329832

B CNGA3 CNGA3 - CDH23 CDH23 -

P CDH23 - CDH23 CNGA3 - CNGA3

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Interaction Database Records

Interaction Experiment Flow

• Make proteins

• Get them together

• Test which ones interact

• Identify proteins that interact

- Where: native vs heterologous host vs in vitro translation vs chemical synthesis

- How much: native level vs overexpressed- Modifications: isoforms, fragments, mutations, PTMs present/absent

C B D

- Where: native organism/cell type/tissue/compartment vs something else- When: cell cycle phase/cell state

- Diverse methods can be used to determine that that proteins interact- Information that can be inferred from each experiment depends on the

method and experimental setup

- Identity of some proteins might be known a priori (eg purified, cloned/tagged bait)

- Identity and/or state of some proteins might be ambiguous (eg unknown splice form, PTMs)

- Some molecules participating in the interaction might remain unidentified

?

F

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Make Proteins

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Identify/Re-identify Proteins That Interacted

Test Which Proteins Interact

Get the Proteins Together

Make Proteins

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Identify/Re-identify Proteins That Interacted

Test Which Proteins Interact

Get the Proteins Together

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

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Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

Interaction Host

Protein Expression

S. cerevisiae

E. coli

in vitro

in vitro

Make Proteins

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Identify/Re-identify Proteins That Interacted

Test Which Proteins Interact

Get the Proteins Together

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

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Make Proteins

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Identify/Re-identify Proteins That Interacted

Test Which Proteins Interact

Get the Proteins Together

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

Interaction Types

B

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Binary Expansion

Make Proteins

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Identify/re-identify Proteins That Interacted

Test Which Proteins Interact

Get the Proteins Together

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

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Protein Identification

Obri et al. Nature 505:648-53 (2014); PMID: 24463511

MS

MassSpec Protein Identification Workflow

UniprotKB: Q15906

• In many cases multiple isoforms exist

• It is hard to unambiguously demonstrate which one is present/absent

• The problem is not unique to MS -similar issues arise when identifying proteins by Western blots

Experimental Ambiguities

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UniprotKB: Q15906

• In some cases there is no single identifier that covers all isoforms

• In some cases there are multiple identifiers that cover the same protein

• Database records change over time

Annotation Ambiguities

Protein Identification

Obri et al. Nature 505:648-53 (2014); PMID: 24463511

UniprotKB: P0C0S8

Gene Duplication

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PICR: Protein Identifier Cross Reference

URL: http://www.ebi.ac.uk/Tools/picr/

• Hosted by EBI (Hinxton, UK)

• Cross-references between close to a 100 of databases (UniProt, Refseq, Ensembl, model organisms, PDB, ...)

• Stable ‘over time’ and over ‘interface’

• Access in a batch mode and as a service (SOAP and RESTful)

• Results presented in either tabular or HTML/XML format

When are two proteins ‘identical’ ?

Possible criteria:

• Identical sequence• Identical species of origin• Identical PTMs• Identical localization (space & time)• Identical gene• Same orthologous group

Human

Cow

Cow Rat

Camel

T4 phage

Definition which proteins are treated as identical is project specific and likely different than the

definition used by at least some authors of the interaction data.

PDB: 3SN6

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Make Proteins

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Identify/re-identify Proteins That Interacted

Test Which Proteins Interact

Get the Proteins Together

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

Interaction RecordInteraction Experiment

Participant List

21

NMolecule Type: Protein (MI:0326)Molecule Name: Cellular tumor antigen p53Molecule Symbol: p53Species of Origin: Human (Taxid:9606)Cross-reference(s): P04637-1 (UniProtKB), NP_00537, (RefSeq), …Experimental Role: Bait (MI:0496)Experimental Source: E.coli K12 (Taxid: 83333)Identification method(s): Predetermined (MI:0396)Features:

…

21

M…Feature Type: Sufficient binding region (MI:0442)

Feature Range: 1-73Identification Method: Deletion analysis (MI:0033)

Interaction Experiment Record

Interaction Detection Method: Pull down (MI:0096)Experiment Host/Cell Line: in vitroInteraction Type: Direct (MI:0407)

Protein Features• Deviations from the default sequence

• Post-translational modifications (PTMs)

• Experimental artifacts

- Mutations (substitutions, deletions, insertions)- Regions necessary/sufficient for binding

- Phosphorylation, methylation, etc- Ubiquitination sites

- Affinity tags (His, FLAG, TAP, GFP, ...)- Labels (biotin, isotope, fluorescent, spin labels,...)- Cross-linking sites- Proteolytic sites

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Molecular Interactions Controlled Vocabulary

Controlled Vocabularies

URL: https://www.ebi.ac.uk/ols/ontologies/mi

Molecular Interactions Controlled Vocabulary

Controlled Vocabularies

URL: https://www.ebi.ac.uk/ols/ontologies/mi

Mutation disrupting interaction

Mutation decreasing interaction

Mutation

Biological feature

Feature type

is a

is a

is a

is a

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Molecular Interactions Controlled Vocabulary

Controlled Vocabularies

Interaction Types

CovalentBinding

Direct Interaction

Physical Association

Association

is a

is a

is a

Enzymatic Reaction

is a