When we conceptualized IMPROViSeD, we envisioned complementing its computational framework with an experimental wet-lab component to validate the software’s predictions. Our goal was to develop an integrated pipeline that could functionally analyze the protein complexes whose structures were elucidated by IMPROViSeD, emphasizing the generation of experimentally derived data to support our computational models.

GPCR Structure-Function Experiments

Initially, we considered studying G-protein coupled receptors (GPCRs), a highly relevant class of proteins involved in signal transduction. GPCRs form one of the largest protein families in human cells and are crucial in various physiological processes by transmitting signals from the extracellular environment to the cell's interior. GPCRs, in their inactive state, form complexes with heterotrimeric G-proteins. Upon ligand binding, the GPCR undergoes a conformational change that activates the G-protein, dissociating it and initiating a signal transduction cascade. [3]

Dysfunctions in GPCR signaling are linked to numerous diseases, such as neurodegenerative disorders and cancer, making them critical targets for therapeutic intervention. We identified three key protein interaction targets for structure-function analysis of GPCRs:

• GPCR-G-protein complex: Interaction between GPCRs and G-proteins in their inactive state. [3]
• β-arrestin complexes: β-arrestin binds to GPCRs, preventing further G-protein activation and leading to receptor desensitization. [4]
• GPCR heteromers: Emerging evidence suggests GPCRs may form heteromers, resulting in distinct functional properties compared to individual subunits. [5]

However, after extensive consideration of the available resources and the experimental demands, we decided that studying GPCRs would not be feasible within our current framework. The complexity of the interactions, coupled with the experimental techniques required, exceeded our capacity at this stage.

The LCN2-MMP9 Complex

As a result, we shifted our focus to exploring the LCN2-MMP9 complex, which plays a significant role in cancer metastasis. Unlike GPCRs, this complex offered a more targeted approach that aligned with both our computational goals and the resources available for experimental validation. Our focus turned to understanding how experimentally generated cross-links could inform the modelling of protein complexes involved in cancer progression, specifically using cross-linking mass spectrometry (XLMS) to generate distance restraints for IMPROViSeD.

LCN2 and MMP9: A Crucial Complex in Cancer Metastasis

Lipocalin-2 (LCN2)

LCN2, also known as neutrophil gelatinase-associated lipocalin, is a 25-kDa protein stored in neutrophil granules. It belongs to the lipocalin family, which specializes in binding and transporting small lipophilic molecules. [6]

LCN2 has several key biological roles:

• Iron trafficking: It captures and transports iron, increasing cytoplasmic levels.
• Immune defense: Released by neutrophils, LCN2 aids in depleting iron from bacterial pathogens.
• Cell differentiation: It influences tissue growth and differentiation by regulating iron-responsive genes.
• Carcinogenesis: LCN2 enhances cancer cell proliferation by facilitating iron uptake in malignant cells. [1]

Matrix Metalloproteinase-9 (MMP9)

MMP9 is an enzyme involved in the breakdown of the extracellular matrix (ECM), playing a vital role in physiological processes like tissue remodelling.

However, in pathological conditions such as cancer, MMP9 contributes to tumor invasion and metastasis by degrading ECM components, thereby enabling cancer cells to migrate to new locations. [1][8]

Role of LCN2-MMP9 Complex in Cancer Metastasis

The interaction between LCN2 and MMP9 prevents the autodegradation of MMP9, enhancing its proteolytic activity and promoting cancer metastasis. This complex is particularly important in cancers of the alimentary canal. By stabilizing MMP9, LCN2 facilitates the invasion and migration of cancer cells. Inhibiting this interaction presents a promising strategy for cancer therapy, but developing effective inhibitors requires detailed structural information about the complex. [1][7]

Past efforts to generate antibodies targeting the LCN2-MMP9 interaction have failed, primarily because the antibodies did not bind to the correct interface. To overcome this challenge, we aim to define the precise binding interface using IMPROViSeD. This requires experimental cross-links between LCN2 and MMP9, which we will generate through XLMS.

Cross-Linking Mass Spectrometry (XLMS)

Overview

XLMS is a powerful technique for identifying interacting regions between proteins by introducing chemical cross-linkers that covalently bind specific amino acids in close proximity. These cross-links provide crucial distance restraints that can be used to model the protein complex's structure. [2]

Methodology

Our approach involves cross-linking LCN2 and MMP9 using Disuccinimidyl Glutarate (DSG), a commonly used cross-linker. The process begins with SDS-PAGE to separate the cross-linked proteins, followed by in-gel digestion using trypsin. The resulting peptides are analyzed by mass spectrometry, and the spectra are compared with non-cross-linked proteins to detect cross-links.

The workflow consists of the following steps:

• Protein Sample: We run SDS-PAGE to separate LCN2 and MMP9 and obtain pure samples.
• In-Gel Digestion: Protein bands are excised and subjected to trypsin digestion to break them into smaller peptide fragments and subjected to mass spectrometry to obtain spectra for non-cross-linked proteins.
• Cross-Linking: The LCN2-MMP9 complex is cross-linked using DSG. Cross-linked peptides are separated by SDS-PAGE to verify the cross-linking, followed by in-gel digestion and mass spectrometry analysis. Peaks unique to the cross-linked peptides are indicative of amino acids involved in the interaction.

Cross-Linking Protocol Optimization

We are currently optimizing the cross-linking protocol to ensure the highest yield of cross-links between LCN2 and MMP9. This involves fine-tuning the DSG concentration and reaction conditions to achieve the most accurate and reproducible results. These cross-links will provide the experimental distance restraints needed for running IMPROViSeD and validating the modelled structures of the LCN2-MMP9 complex.

Protocols

Solutions required

1. ABC (Ammonium Bicarbonate) 200mM stock - 5ml
2. ABC 50mM - 10ml

   2.5 ml stock ABC solution + 7.5 ml MilliQ H₂O

3. 100% ACN (acetonitrile)
4. 50mM ABC in 50% ACN - 10ml

   2.5ml ABC from 200 mM stock + 2.5 ml MilliQ water + 5 ml ACN

5. 1M stock DTT (Dithiothreitol) - 1ml

   154 mg DTT + 1 ml H₂O

6. 10mM DTT - 1ml

   10 µl DDT from 1M stock + 990 µl of 50mM ABC solution

7. IAA- 10mg/ml in 50mM ABC - 1ml
8. 20 ng /μl

In Gel Digestion

1. Excise the band from de-stained SDS PAGE gel (after coomassie brilliant blue staining). Chop the band into small pieces.
2. Add solution 4 (50mM ABC in ACN) enough to soak the gel pieces (~100μl). Incubate at 37°C for 10 min. Centrifuge at 1000 rpm for 5 min and discard the supernatant.
3. Add 100% ACN (~100 μl). Vortex and short spin to remove ACN. Repeat steps 2 and 3 until gel pieces are completely transparent.
4. Add 10mM DTT (~100 μl). Incubate at 56°C for 45 min. Centrifuge at 1000 rpm and discard the supernatant.
5. Wash with 100% ACN, vortex, short spin, and remove supernatant. Speed vacuum until dry at 30°C. Keep in ice for 10 min.
6. Add 20ng/μl Trypsin in a 1:20-1:50 ratio. Top up with the 50mM ABC solution if gel pieces are not submerged. Chill on ice for 10 min so that the gel soaks up the trypsin.
7. Incubate at 37°C for 16 hrs.

Peptide extraction

Extraction solution: 60% ACN (600μl) + 5% Formic acid (50μl) + H₂O (300μl) - 1ml

1. Take supernatant in fresh MCT. Do not discard gel pieces.
2. Add peptide extraction solution to gel pieces (enough to soak). Vortex for 5 min, centrifuge at 1400 rpm for 15 min. Add supernatant to initial MCT with trypsin-digested peptide.
3. Repeat step 2 (3-4 times).
4. Speed vacuum at 30˚C till dry (no parafilm).
5. Store at -20˚C for mass spectrometry to be done.

References

1. Lin CW, Tseng SW, Yang SF, Ko CP, Lin CH, Wei LH, Chien MH, Hsieh YS. Role of lipocalin 2 and its complex with matrix metalloproteinase-9 in oral cancer. Oral Dis. 2012 Nov;18(8):734-40.
2. Graziadei A, Rappsilber J. Leveraging crosslinking mass spectrometry in structural and cell biology. Structure. 2022 Jan 6;30(1):37-54.
3. Digby GJ, Lober RM, Sethi PR, Lambert NA. Some G protein heterotrimers physically dissociate in living cells. Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17789-94.
4. Tobin AB. G-protein-coupled receptor phosphorylation: where, when and by whom. Br J Pharmacol. 2008 Mar;153 Suppl 1(Suppl 1):S167-76.
5. Dale NC, Johnstone EKM, Pfleger KDG. GPCR heteromers: An overview of their classification, function and physiological relevance. Front Endocrinol (Lausanne). 2022 Aug 30;13:931573.
6. Kjeldsen L, Johnsen AH, Sengeløv H, Borregaard N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem. 1993 May 15;268(14):10425-32. PMID: 7683678.
7. Candido S, Abrams SL, Steelman LS, Lertpiriyapong K, Fitzgerald TL, Martelli AM, Cocco L, Montalto G, Cervello M, Polesel J, Libra M, McCubrey JA. Roles of NGAL and MMP-9 in the tumor microenvironment and sensitivity to targeted therapy. Biochim Biophys Acta. 2016 Mar;1863(3):438-448.
8. Vandooren J, Van den Steen PE, Opdenakker G. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): the next decade. Crit Rev Biochem Mol Biol. 2013 May-Jun;48(3):222-72.