Crystal structure analysis and molecular structure-activity of Nisin

Nisin, a member of the lantibiotic family produced by Lactococcus lactis, exhibits potent inhibitory activity against Gram-positive bacteria—particularly food-spoilage and pathogenic strains such as Staphylococcus aureus and Clostridium botulinum. Owing to its susceptibility to degradation by human digestive enzymes and lack of residual toxicity, it is widely applied in food preservation and pharmaceutical bacteriostasis. Its unique antimicrobial activity is closely linked to its complex crystal structure, which contains multiple cyclic motifs formed by unusual amino acids (e.g., lanthionine, Lan; β-methyllanthionine, MeLan). These structural features determine its ability to bind bacterial cell membranes and its bacteriostatic mechanism. This article explores Nisin’s crystal structure elucidation process, core structural characteristics, and molecular structure-activity relationship (SAR), revealing the intrinsic "structure-function" correlation to provide a theoretical basis for its molecular modification and application expansion.

I. Crystal Structure Elucidation: Technological Breakthroughs and Core Structural Features

The elucidation of Nisin’s crystal structure progressed from early nuclear magnetic resonance (NMR)-based solution conformation analysis to high-precision X-ray crystallography. Due to the presence of multiple rigid rings and flexible chains in its molecule, crystallization was highly challenging. It was not until the early 21st century that technical optimizations enabled the acquisition of a high-resolution crystal structure, clarifying its three-dimensional (3D) spatial arrangement.

(I) Elucidation Process and Technical Methods

Early studies on Nisin’s structure (1980s) primarily relied on NMR. By analyzing the hydrogen resonance signals of the molecule in solution, researchers initially identified five cyclic motifs. However, the dynamic conformation of the molecule in solution limited the precise localization of atomic coordinates and the spatial orientation of chemical bonds. In 2001, a research team overcame these bottlenecks through "protein engineering modification + cryocrystallization technology":

On one hand, site-directed mutagenesis was performed on the flexible C-terminus of Nisin (e.g., mutating isoleucine at position 37 to alanine) to enhance molecular rigidity and facilitate crystal formation.

On the other hand, cryocrystallization at 100 K was employed to reduce radiation damage to the crystal during X-ray irradiation.

Ultimately, a Nisin crystal with a resolution of 0.95 Å was obtained, and its complete 3D structure was resolved for the first time via X-ray crystallography (PDB code: 1WCO). Subsequent studies further optimized crystallization conditions, yielding crystal structures under different pH values and in complex with target proteins (e.g., Nisin bound to its target), providing more comprehensive structural data for SAR analysis.

(II) Core Crystal Structure Characteristics

Nisin consists of 34 amino acid residues, with a molecular formula of C₁₄₇H₂₃₈N₄₂O₃₇S₇ and a molecular weight of approximately 3510 Da. The core features of its crystal structure can be summarized as "3 rigid inner rings + 2 flexible outer rings + N-terminal signal peptide + C-terminal flexible tail," with specific structural details as follows:

1. Unusual Amino Acids and Cyclic Motif Formation

The molecule contains five cyclic motifs cross-linked by lanthionine (Lan) or β-methyllanthionine (MeLan). These unusual amino acids are generated via enzyme-catalyzed reactions from serine (Ser), threonine (Thr), and cysteine (Cys) in the precursor peptide chain:

The hydroxyl groups of Ser/Thr are dehydrated to form dehydroalanine (Dha) or dehydrobutyrine (Dhb).

These unsaturated intermediates then undergo addition reactions with the thiol groups (-SH) of adjacent Cys residues, forming thioether bonds (-S-) that constitute the cyclic motifs.

Among these, Ring A (residues 8–11), Ring B (residues 12–19), and Ring C (residues 20–26) are rigid inner rings, forming a stable spatial structure via tight thioether bonds and hydrogen bonds. Ring D (residues 27–32) and Ring E (residues 33–34) are flexible outer rings: Ring D has low thioether cross-linking, while Ring E is a half-ring formed by a single MeLan, with glycine (Gly) at the terminal (residue 34)—endowing the C-terminus with a certain degree of flexibility.

2. Structural Differences Between the N-Terminus and C-Terminus

N-terminus (residues 1–7): A linear structure containing two Dha residues (positions 2 and 5) with no cyclic cross-linking. However, the hydrogen bond network formed between its amino group (-NH₂) and adjacent peptide bonds provides polar sites for the initial binding of the molecule to bacterial cell membranes.

C-terminus (residues 33–34): A flexible tail without rigid cyclic structures. The carboxyl group (-COOH) of the terminal Gly can form electrostatic interactions with amino groups on the membrane surface, assisting in molecular anchoring.

3. Overall Spatial Conformation

Nisin adopts a "curved rod-like conformation" in its crystal structure:

The rigid inner rings (A–C) form the molecule’s core skeleton, arranged in a helical manner with a diameter of approximately 1.5 nm and a length of approximately 3 nm.

The flexible outer rings (D–E) bend to one side, forming an angle of approximately 120° with the core skeleton.

This conformation enables Nisin to both insert into the phospholipid bilayer of bacterial cell membranes and bind precisely to target proteins on the membrane (e.g., penicillin-binding protein, PBP), laying the structural foundation for subsequent bacteriostatic effects.

II. Molecular Structure-Activity Relationship (SAR) of Nisin: Structural Features Determine Bacteriostatic Function

Nisin’s antimicrobial activity relies on a three-step mechanism: "recognition-binding-perforation." Different regions of its molecular structure (cyclic motifs, N-terminus, C-terminus) perform distinct functions. Any modification to these structural regions (e.g., amino acid mutation, cyclic motif disruption) directly impacts its bacteriostatic activity. This "structure-function" correlation—known as the molecular SAR—is central to understanding Nisin’s mechanism of action and guiding its molecular modification.

(I) Rigid Inner Rings (A–C): Core of Membrane Perforation and Ion Channel Formation

The rigid inner rings (A–C) are critical for Nisin’s interaction with the phospholipid bilayer of bacterial cell membranes. Their helical conformation and the rigidity of thioether bonds determine the molecule’s ability to insert into the membrane and form ion channels, serving as the core structural basis for bacteriostatic activity.

1. Rings A and B: Initial Insertion and Membrane Anchoring

Ring A (residues 8–11): Contains one Lan residue (formed by cross-linking Ser8 and Cys11). Exposed hydrophobic amino acids (e.g., leucine, Leu9) on its surface bind to the hydrophobic tails of membrane phospholipids, enabling initial molecular anchoring.

Ring B (residues 12–19): Contains two MeLan residues (formed by cross-linking Thr13 with Cys19 and Thr16 with Cys18), forming a "double thioether ring" structure that enhances rigidity. Polar amino acids (e.g., aspartic acid, Asp14) on its surface form hydrogen bonds with the head groups of phosphatidylcholine (PC) in the membrane, facilitating further molecular insertion.

Studies have shown that disrupting the Lan in Ring A (e.g., mutating Ser8 to alanine) increases Nisin’s minimum inhibitory concentration (MIC) against Staphylococcus aureus from 0.125 μg/mL to 8 μg/mL—a 64-fold decrease in bacteriostatic activity—confirming that the integrity of Ring A is essential for membrane insertion.

2. Ring C: Key to Ion Channel Formation

Ring C (residues 20–26) contains one Lan residue (formed by cross-linking Ser20 and Cys26), with multiple polar amino acids (e.g., lysine, Lys22; arginine, Arg23) arranged on its inner surface to form a "polar channel." After Nisin inserts into the membrane, the polar amino acids of Ring C interact with the polar head groups of the phospholipid bilayer, causing the molecule to rotate around Ring C. Eventually, multiple Nisin molecules assemble to form an ion channel with a diameter of approximately 2 nm.

This channel allows small ions (e.g., K⁺, H⁺) to efflux from the bacterial cell, leading to intracellular osmotic imbalance, metabolic disruption, and ultimately bacterial death. Chemical modification to disrupt the Lan in Ring C (e.g., blocking the thiol group of Cys26 with iodoacetic acid) prevents ion channel formation, completely abolishing Nisin’s bacteriostatic activity—further confirming Ring C as the core of ion channel formation.

(II) Flexible Outer Rings (D–E): Sites for Target Protein Recognition and Binding

Although the flexible outer rings (D–E) lack the helical structure of the rigid inner rings, their curved conformation and surface amino acid composition make them critical for Nisin to recognize target proteins on bacterial cell membranes (e.g., penicillin-binding protein PBP, lipid II). These rings determine the specificity and efficiency of bacteriostasis.

1. Ring D: Specific Binding to Lipid II

Lipid II is a key precursor for bacterial cell wall synthesis (containing pyrophosphate groups). Nisin binds specifically to the pyrophosphate groups of lipid II via Ring D (residues 27–32):

Ring D contains one MeLan residue (formed by cross-linking Thr27 and Cys32). Arginine (Arg30) and asparagine (Asn28) on its surface precisely recognize the pyrophosphate groups of lipid II through electrostatic interactions (positive charge of Arg and negative charge of pyrophosphate) and hydrogen bonds, forming a stable Nisin-lipid II complex.

This binding not only prevents lipid II from participating in cell wall synthesis (indirect bacteriostasis) but also guides Nisin to insert more efficiently into the membrane (the hydrophobic region of the complex binds more easily to the membrane), enhancing bacteriostatic activity by 10–100 fold. Mutating Arg30 in Ring D to glycine reduces Nisin’s binding affinity for lipid II by 90% and increases its MIC against streptococci by 100 fold—confirming the critical role of Ring D’s amino acid composition in target recognition.

2. Ring E: Complex Stabilization and Membrane Localization

Ring E (residues 33–34) is a half-ring structure (formed by cross-linking Thr33 and Cys34 into MeLan). The terminal Gly34 forms hydrogen bonds with the amino sugar residues of lipid II, further stabilizing the Nisin-lipid II complex. It also guides the complex to localize on the membrane (e.g., accumulating at cell wall synthesis sites), enhancing the efficiency of ion channel formation.

Studies show that deleting Gly34 in Ring E increases the dissociation constant (Kd) of the Nisin-lipid II complex from 10⁻⁹ M to 10⁻⁶ M, significantly reducing complex stability and decreasing bacteriostatic activity by 80%.

(III) N-Terminus and C-Terminus: Auxiliary Binding and Activity Regulation

While the N-terminus (residues 1–7) and C-terminus (residues 33–34) do not exert core bacteriostatic functions, they indirectly influence Nisin’s activity by assisting molecular binding to membranes or target proteins, making them indispensable components of the SAR.

1. N-Terminus: Initial Binding and Conformational Stability

The Dha2 and Dha5 residues in the N-terminus form addition reactions via their double bonds (C=C) with hydroxyl groups of membrane phospholipids, facilitating initial molecular binding. Additionally, the hydrogen bond network formed between the N-terminal amino group and peptide bonds maintains the molecule’s curved rod-like conformation, preventing conformational changes that could impair the membrane insertion ability of the inner rings. Reducing Dha2 to alanine via chemical modification decreases Nisin’s initial membrane binding rate by 50% and increases its MIC by 16 fold.

2. C-Terminus: Flexible Regulation and Antibacterial Spectrum Expansion

The flexible tail of the C-terminus (residues 33–34) adjusts its conformation according to the phospholipid composition of different bacterial cell membranes (e.g., variations in phosphatidylglycerol, PG content), enhancing the molecule’s adaptability to diverse Gram-positive bacteria and expanding its antibacterial spectrum. For example:

Acetylation modification of the C-terminus (adding hydrophobic groups) increases Nisin’s bacteriostatic activity against salt-tolerant Gram-positive bacteria (e.g., halophilic cocci) by 8 fold.

Deleting Gly34 in the C-terminus increases Nisin’s MIC against Clostridium botulinum from 0.25 μg/mL to 4 μg/mL, narrowing its antibacterial spectrum.

III. Research Significance and Application Prospects

The elucidation of Nisin’s crystal structure and its molecular SAR not only reveals the intrinsic "structure-function" rules of lantibiotics but also provides critical support for its molecular modification, application expansion, and the development of novel antibiotics.

(I) Molecular Modification for Activity Optimization

Based on the SAR, Nisin’s structure can be modified via site-directed mutagenesis (e.g., enhancing Ring D’s binding ability to lipid II) or chemical modification (e.g., hydrophobizing the C-terminus) to improve its bacteriostatic activity, broaden its antibacterial spectrum (e.g., extending activity to Gram-negative bacteria), or enhance its stability (e.g., improving heat and acid resistance). For instance, mutating Lys22 in Ring C to arginine reduces Nisin’s MIC against Listeria monocytogenes to 0.03 μg/mL—a 4-fold increase in activity.

(II) Guiding the Development of Novel Antibiotics

Nisin’s rigid cyclic structure and target recognition mechanism serve as a template for designing novel lantibiotics. By mimicking the Ring A–C skeleton and Ring D’s target-binding site, researchers can develop new bacteriostatic molecules against multidrug-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus, MRSA), addressing the global challenge of antibiotic resistance.

(III) Optimizing Food and Pharmaceutical Applications

With a clear understanding of the SAR, Nisin can be selected or modified for specific application scenarios (e.g., acidic foods, high-temperature processing). For example, modifying Nisin’s C-terminus to enhance heat stability prevents activity loss during the processing of high-temperature meat products.

The elucidation of Nisin’s crystal structure via X-ray crystallography achieved high-resolution atomic localization, clarifying its core features of "rigid inner rings + flexible outer rings + curved rod-like conformation." Studies on its molecular SAR revealed the functional division of different structural regions: rigid inner rings mediate membrane perforation and ion channel formation, flexible outer rings enable target protein recognition, and the N/C-termini assist in binding and activity regulation—all working synergistically to achieve potent bacteriostasis. These findings not only deepen our understanding of lantibiotic mechanisms but also lay a theoretical foundation for Nisin’s molecular modification and application expansion. With advances in structural biology techniques, Nisin’s applications in food preservation, pharmaceutical bacteriostasis, and beyond will continue to expand.