LL-37: Molecular Structure

Molecular Structure and Properties#

LL-37 is a peptide whose molecular structure and properties have been characterized through analytical chemistry and structural biology studies.

Amino Acid Sequence#

Overview LL-37 is the 37–amino acid, C‑terminal antimicrobial peptide released from the human cathelicidin precursor hCAP18 (CAMP) by proteolytic processing (notably by proteinase 3 in neutrophils). It is linear (no disulfide bonds), cationic, and amphipathic. In aqueous buffers it is largely unstructured, but it adopts an α‑helical, amphipathic conformation in membrane‑mimetic environments, where it can oligomerize into dimers and tetramers that form conductive channels; higher‑order fibrillar assemblies are also observed under membrane/detergent conditions.

Amino acid sequence and origin • Sequence (37 aa): LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES. • Molecular origin: C‑terminal peptide derived from the hCAP18 propeptide; excision by proteinase 3 generates mature LL‑37.

Physicochemical properties • Length and mass: 37 residues; reported molecular mass ≈ 4.5 kDa (4493.4 Da). • Net charge at physiological pH: +6 at pH ~7–7.4, reflecting a high content of Lys/Arg balanced by acidic residues. • Isoelectric point: literature reports a theoretical pI ~11 for LL‑37, consistent with its cationic composition. • Charge distribution: Approximately 16 of 37 residues are charged; the amphipathic helix presents a polar/cationic face (enriched in Lys/Arg) and a hydrophobic face. Post‑translational deimination (citrullination) of Arg reduces positive charge and attenuates antibacterial activity, underscoring charge dependence. • Amphipathicity and representative faces: The hydrophobic (concave) face includes L2, F5, F6, I13, F17, I20, V21, I24, F27, L28, L31; the opposing face is polar/cationic.

Structural features • Secondary structure in solution versus membranes: Unstructured or partially helical in aqueous solution; in anionic or zwitterionic micelles/bilayers LL‑37 adopts an α‑helix spanning roughly residues 2–31 or 4–33, with disordered termini. • Helical architecture and motifs: A helix–break–helix arrangement with a bend around residues 14–16 is recurrent. Intrahelical salt bridges (e.g., K12–E16; possibly K8–E11) and a hydrophobic cluster involving I13, F17, I20 stabilize the fold and membrane engagement. • Oligomerization and channels: LL‑37 is in equilibrium between monomer and oligomers in solution; in membrane mimics it forms antiparallel dimers and an asymmetric tetramer that assembles a narrow, positively charged channel with small but defined conductance in planar lipid membranes. MD simulations support channel stability and water permeation; aromatic rings (F17, F27) form girdles that may aid insertion. LL‑37 can further assemble into head‑to‑tail fibers/nanofibrils on membranes. • Amphipathic membrane topology: In micelles, the hydrophobic face is buried toward the micelle interior while the hydrophilic face is solvent‑exposed, consistent with surface‑bound adsorption and curvature matching.

Available structures and identifiers NMR/X‑ray structures capture monomers, dimers, and higher assemblies in detergents or in detergent‑free crystals. Reported entries include: 2K6O (micelle NMR), 5NMN (monomer in DPC), 5NNM (detergent‑free dimer), 5NNT (DPC dimer), 5NNK (LDAO dimer), and 7PDC (tetrameric channel); fragment structures include LL‑37(17–29) (2FBS) and an N‑terminal fragment (2FBU).

Embedded summary table

Notes on parameter sensitivity and modifications Electrostatics are central to activity: increases or decreases in cationic content modulate pI and net charge, altering LPS binding and antibacterial function. For example, citrullination reduces the net positive charge and diminishes killing despite largely preserved helicity. The balance of hydrophobic and cationic residues and the ability to oligomerize at membranes underpin channel formation and membrane disruption.

References (citation IDs) Sequence, charge and origin:. Secondary structure and amphipathicity:. Oligomerization/channels and fibers:. Mass and pI:.

Stability and Formulation#

We synthesize current evidence on LL-37 stability across pH, temperature, proteolysis, and formulation, emphasizing quantitative findings and practical implications.

pH-dependent stability and matrix effects. LL-37 is largely unstructured in pure water but rapidly forms an α-helix in low-ionic-strength physiological buffers; at low pH (≈2–5) its conformation shifts toward unstructured, and endosomal pH (~6) can trigger dissociation from electrostatically bound cargos. In PBS, only about 20–30% of LL-37 is α-helical, and physiological Na+ (~100 mM) reduces antimicrobial activity by ≈2–8-fold. Polyanions in biological matrices strongly sequester/inactivate LL-37: plasma glycosaminoglycans (GAGs) at ≥1 μg/mL inhibit activity, and 25–50 μg/mL GAGs in wound settings can abolish activity of 100 μM LL-37; apoA-I/lipoproteins bind LL-37 with Kd ~1–2 μM, substantially scavenging peptide and reducing activity. Citrullination reduces net positive charge and lowers binding to negatively charged lipids. Together, ionic strength, polyanions, and charge-modifying PTMs govern functional stability in situ.

Temperature sensitivity. Circular dichroism thermal melts quantify concentration-dependent thermal stability. Native LL-37 has melting temperatures (Tm) of ≈69°C at 100 μM, ≈55°C at 50 μM, and ≈47°C at 15 μM. A pentacitrullinated variant exhibits slightly higher Tm values (≈73°C, ≈67°C, and ≈54°C at the same concentrations). Both native and citrullinated LL-37 unfold to random coil at 95°C and refold upon cooling to 25°C, indicating reversible thermal unfolding under these conditions.

Proteolytic degradation pathways and post-translational modifications. LL-37 is susceptible to both host and microbial proteases. In vitro assays at 37°C (e.g., peptide ~136 μM; peptide:enzyme ~300:1; 4 h) map cleavage preferences for hydrophobic P1 residues. Key proteases include Pseudomonas aeruginosa elastase (PE), Staphylococcus aureus aureolysin and V8 protease, and host human neutrophil elastase (HNE); these enzymes readily degrade LL-37 and internal segments like EFK17. Clinically, chronic wound fluids with abundant bacterial and host proteases contribute to LL-37 inactivation. The propeptide is processed to LL-37 by proteinase 3 in vivo. Post-translationally, PAD2/PAD4-catalyzed citrullination of arginines decreases cationicity, impairs bactericidal and immunomodulatory functions, alters lipid/LPS binding, and produces species with distinct modification patterns; N-/C-terminal capping can reduce proteolysis by specific staphylococcal proteases. These mechanisms underline the short functional half-life of LL-37 in protease-rich milieus.

Formulation considerations to improve stability. Nanostructured carriers and dressings can mitigate proteolysis and modulate release. Post-loaded cubosomes shield LL-37 from PE and HNE, yielding increased antibacterial effect after enzyme exposure. Release is front-loaded over the first 6 hours, and physiological salt markedly slows release; in one condition only ~30% of peptide was released by 24 hours in salt-containing medium. However, carrier association can shift the activity spectrum (LL-37-loaded cubosomes favored Gram-negative activity). More broadly, liposomes, polymeric nanoparticles (e.g., PLGA), chitosan-based systems, hydrogels, and protease-absorbing dressings increase local residence, reduce enzymatic access, and can preserve function in protease-rich wounds. Excipients that provide steric protection (e.g., P407 at cubosome rims) and matrices that limit polyanion sequestration are advantageous; testing under relevant ionic strength and protease conditions is essential.

Practical takeaways. LL-37’s functional stability decreases at low pH, high ionic strength, and in polyanion-rich, protease-rich environments. Thermal unfolding is reversible, with Tm rising at higher peptide concentration and modestly with citrullination, but proteolysis remains the dominant inactivation pathway in vivo. Formulations such as cubosomes and other nano/macrocarriers, plus wound dressings that remove proteases, can substantially improve stability and activity duration; release kinetics and ionic-strength effects must be optimized for the intended site of use.

Pharmacokinetics#

We synthesized the pharmacokinetic properties of the human cathelicidin LL-37 from primary imaging PK in mice using a radiolabeled analogue and from reviews addressing systemic bioavailability and proteolysis. A structured summary is provided below, followed by a narrative synthesis.

Absorption

• Intravenous: Following a 1 mg/kg IV bolus in mice, blood Tmax was 0.1 h, indicating immediate systemic availability; however, circulating exposure was limited as a large fraction rapidly redistributed to tissues, notably liver and lungs.
• Subcutaneous: After 1 mg/kg SC dosing in mice, LL-37 was well absorbed from the injection site but with notable depot retention. Absorption from the SC site was biphasic with a fast half-life of 1.7 h and a slow half-life of 54.2 h; blood Cmax ≈1.1 ± 0.1 μg/mL occurred at ~4 h. Injection-site content declined from ~28% ID at 4 h to ~6.6% ID at 48 h.
• General/other routes: Reviews emphasize that systemic absorption of unmodified LL-37 is poor for oral and many extravascular routes because of enzymatic degradation and low permeability; topical delivery is commonly favored.

Distribution

• Early pulmonary uptake: IV dosing produced high, patchy lung uptake at 0.1 h (~10% of dose; focal areas >20 g/mL), which diminished by 2 h.
• Hepatic and splenic uptake: The liver dominated distribution after IV dosing, holding ~50–70% of dose throughout the study; at 48 h, liver held 47.0 ± 0.9% ID/organ (39.7 ± 1.1% ID/g). Spleen concentrations were 21–27% ID/g with ~1.5% ID/organ.
• Skeletal accumulation: Gradual accumulation in bone and joints over time was observed for both routes; at 48 h post-IV, bone held ~9.4 ± 1.3% ID/organ; after SC, bone held ~13.7% ID/organ outside the injection site.
• Blood exposure: IV blood levels were modest initially (4.3 ± 1.5 μg/mL at 0.1 h) and fell below 1 μg/mL after ~7 h. After SC, blood exposure was sustained by continued absorption, with MRT0-∞ ≈18.6 h.

Metabolism/Proteolysis

• Direct in vivo proteolytic products were not quantified in the imaging study. Reviews document that LL-37 is susceptible to proteolysis by host and bacterial proteases (e.g., S. aureus aureolysin cleaving the core 13–32 region), and plasma protein binding can reduce free circulating peptide. These mechanisms underlie poor systemic stability and bioavailability and motivate chemical/formulation strategies to enhance protease resistance.

Elimination and Clearance Pathways

• IV: Predominantly hepatic clearance with a minor renal component. Kidneys and urine peaked early (kidney 1.5 ± 0.1 g/mL at 0.1 h; urine 3.5 ± 0.3 g/mL at 0.1 h), but the cumulative urinary fraction was small (1–3% ID early), and slow excretion into urine and feces continued through 48 h.
• SC: Once absorbed, clearance involved kidney, liver, and spleen. Urine showed a spike at 4 h (2.6 ± 0.4 g/mL; 11.5 ± 1.6% ID/g), coinciding with the faster absorption phase, with ongoing slow excretion thereafter.

Half-life

• Whole-body (imaging-based): IV one-phase whole-animal decay t1/2 ≈115.3 h; only ~25% of the injected dose was eliminated by 48 h. SC whole-body decay was biphasic with fast t1/2 ≈0.7 h and slow t1/2 ≈68.3 h; ~49% of the dose remained at 48 h.
• Blood exposure: IV blood PK showed Tmax 0.1 h, AUC0-∞ 28.2 ± 7.5 μg·h/mL, MRT0-∞ 10.8 ± 2.3 h; after SC, AUC0-∞ 63.1 ± 11.8 μg·h/mL and MRT0-∞ 18.6 ± 2.3 h. Reviews describe short plasma half-lives for unmodified peptides generally; some secondary sources cite ~1.16 h plasma t1/2 in rats, but primary LL-37-specific rat PK numbers were not verified in our sources.

Bioavailability

• Absolute SC bioavailability relative to IV was not reported in the imaging study. Reviews consistently state that oral and systemic bioavailability of unmodified LL-37 is low due to enzymatic degradation and permeability constraints; topical administration is typically preferred without protective formulation.

Interpretation and caveats

• The quantitative mouse data derive from a radiometal-labeled LL-37 (67Ga-NOTA-LL-37). The chelate was attached to the N‑terminus and was shown to be stable in vitro, and the labeling conditions preserved peptide size on SDS-PAGE, supporting its use as a surrogate. Nonetheless, radiolabeling and species-specific physiology can influence apparent distribution and elimination. The study provides robust relative organ distribution, elimination route evidence, and blood exposure metrics over 48 h but does not directly measure proteolytic metabolites or absolute bioavailability. Reviews support that proteolysis and plasma binding underlie the low systemic exposure and bioavailability of native LL-37.

Related Reading#

• LL-37 overview and research guide
• Peptides similar to LL-37
• LL-37 research evidence