MACS
Evidence Matrix

EN TR

MACS Research Documentation

Mechanistic Evidence Matrix

Project
MACS
Document type
Technical Documentation
Year
2026

Cross-references each mechanistic claim with structural, dynamic, energetic, quantitative, and uncertainty evidence.

M2

B293 changes between alternative polar microenvironments

Moderate
Structural Evidence

High-pKa state places B293 near B289; low-pKa state places B293 near the B128 backbone oxygen

Dynamic / Network Evidence

Contact–pKa and distance time-series analyses support state-dependent local rearrangement

Energetic / pKa Evidence

Ensemble PROPKA identifies an unusually high and heterogeneous B293 pKa

Key Quantitative Result B293 mean pKa = 8.6708 ± 0.689; fraction pKa >9 = 0.337; selected contacts: B293 ND1–B289 O = 2.715 Å; B293 ND1–B289 OD2 = 3.285 Å; B293 NE2–B128 O = 2.762 Å Main Uncertainty PROPKA is an empirical structure-based estimator; representative frames do not define full populations
M3

B324 exhibits strong pKa-coupled microenvironment remodeling

Moderate to strong
Structural Evidence

High-pKa B324 state forms close contacts with B285 GLU; lower-pKa states adopt different local arrangements

Dynamic / Network Evidence

Distance and contact analyses link B324 environment to conformational state

Energetic / pKa Evidence

Ensemble PROPKA shows broad pKa heterogeneity and strong pH-sensitive protonation propensity

Key Quantitative Result B324 mean pKa = 7.7672 ± 1.227; fraction pKa 6–8 = 0.752; fraction pKa >9 = 0.188; selected high-state contacts: ND1–B285 OE1 = 2.719 Å and ND1–B285 OE2 = 2.897 Å Main Uncertainty Large pKa dispersion; structural-state populations require explicit population analysis or constant-pH MD
M4

Module B is more compact and internally contacted at pH 6.8

Moderate
Structural Evidence

Average and representative structures indicate altered Module B organization

Dynamic / Network Evidence

Module B contacts, radius of gyration, and shape descriptors consistently differ between conditions

Energetic / pKa Evidence

No direct free-energy proof; interpretation is based primarily on conformational metrics

Key Quantitative Result Last 100 ns: contacts 778.09 ± 13.89 at pH 6.8 vs 748.63 ± 14.51 at pH 7.4; Rg 2.6284 ± 0.0159 nm vs 2.6481 ± 0.0186 nm; asphericity 1.42259 ± 0.07168 nm² vs 1.60608 ± 0.11409 nm²; kappa² 0.09890 ± 0.00345 vs 0.06252 ± 0.00704 Main Uncertainty Only one trajectory per pH; metric differences may include slow conformational drift and are not independent replicates
M5

The histidine-rich regions reorganize dynamically between pH conditions

Moderate to strong
Structural Evidence

Clean histidine-network views place selected pH-sensitive histidines within the global architecture

Dynamic / Network Evidence

ANM, DCCM, and network analyses identify condition-dependent changes in fluctuation and communication patterns

Energetic / pKa Evidence

Ensemble PROPKA identifies several histidines with predicted pKa values within or near the physiological switching range

Key Quantitative Result A275 Δfprot = 0.1321; A282 = 0.1871; A289 = 0.2750; A296 = 0.2365; B324 = 0.2250; B329 = 0.2503; B332 = 0.1996; ANM fluctuations near A293–A296 are higher at pH 7.4 Main Uncertainty Average-structure visualization lacks side-chain detail; ANM is coarse-grained; PROPKA does not model coupled titration explicitly
M6

Global A–B PB-MMPBSA energy is directionally more favorable at pH 6.8

Moderate directional
Structural Evidence

Global overlays show substantial conformational divergence between trajectory-derived averages

Dynamic / Network Evidence

DCCM and intermodule network analyses indicate altered coupling between conditions

Energetic / pKa Evidence

PB-MMPBSA endpoint calculations were completed using 1001 frames per pH condition; configurational entropy was not included

Key Quantitative Result ΔTOTAL pH 6.8 = +61.43 kcal/mol (SD 59.38; SEM 1.88); ΔTOTAL pH 7.4 = +71.15 kcal/mol (SD 41.03; SEM 1.30); pH 6.8 − pH 7.4 = −9.72 kcal/mol. Component differences: ΔEEL = −315.54, ΔEPB = +225.92, ΔGGAS = −113.00, ΔGSOLV = +103.28 kcal/mol Main Uncertainty One trajectory per pH, correlated frames, no configurational entropy, large framewise dispersion, and no replicate-level uncertainty; the reported framewise SEM should not be interpreted as independent replicate significance