The activating receptor NKp30 is important in NK cell killing of cancer cells. Here, we demonstrated that a pair of splice signals in the Ig domain exon of human NKp30 is largely conserved among primates and placental mammals and produces an alternatively spliced NKp30 ectodomain (NKp30-S) with an in-frame, nonartefactual deletion of 25 amino acid residues. Transfection yielded NKp30-S bands in NKL cells but not in 293T cells, suggesting that the splicing mechanism is cell-specific. Molecular modeling indicated that the overall folding of NKp30-S is maintained compared with NKp30. NKp30-S was expressed at the cell surface but did not bind to the NKp30 ligand B7H6 in soluble fusion protein or reporter cell assays. Single-cell RNA sequencing showed that NKp30-S transcription was not restricted to major NK cell subsets but suggested that a small fraction of blood NK cells expressed only the NKp30-S splice variant. The ratio of NKp30-S to full-length transcript was increased after activation of blood NK cells with IL-2 or IL-15 or crosslinking with anti-CD16 antibody, suggesting that this unique splicing mechanism, not seen in other Ig superfamily proteins, is regulated and may play a role in modulating NK cell responsiveness toward B7H6+ cancer cells in vivo.

The human gut microbiome carries a large array of biosynthetic gene clusters (BGCs) that encode the production of secondary metabolites, yet their temporal dynamics and role during microbial colonization remain largely unexplored. Here, we tracked BGCs profile over time in a cohort of healthy adults, and identified two distinct groups: persistent, which are stable over time, and transient, which are more sporadic. Functional annotations indicated persistent gene clusters are enriched in antibiotic resistance mechanisms, while transient ones more frequently carry virulence-associated genes. We then examined colonization of these two groups in the context of fecal microbiome transplantation. Our results show that persistent gene clusters exhibit higher colonization rates than transient ones. These findings contribute to our understanding of how microbial metabolites influence host health, potentially guiding future therapeutic strategies targeting the microbiome.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a central regulator of immune signalling, yet how its conformational dynamics govern activation remains poorly understood. Here, we integrate NMR relaxation measurements, molecular dynamics simulations, and ensemble modelling to characterise the solution-state behaviour of the catalytic core MALT1(PCASP-Ig3)339-719 under different ionic conditions. Under low-salt conditions, all simulations converge to a dominant inactive ensemble characterised by inward rotation of residue W580 and coordinated rearrangements of regulatory loops, indicating that the inactive state is energetically favoured in solution. At intermediate ionic strength, reversible loop motions permit transient access to active-like conformations, whereas high-salt conditions suppress loop dynamics and trap the protein in its functional starting conformational basin. Comparison with experimental NMR relaxation data identifies the low-salt inactive ensemble as the best representation of solution-state dynamics, revealing stable hydrophobic cores and loop-localised conformational plasticity. Together, these results support ionic strength as a key determinant for MALT1 conformational equilibria in solution and suggest how coordinated loop dynamics regulate access to catalytically competent states. This provides a dynamic framework for future structure-based modulation of MALT1 activity.

The interaction between a class I peptide-major histocompatibility complex (pMHC) and a T cell receptor (TCR) plays a central role in the elicitation of CD8+ T cell immune responses. As a result, considerable effort has been invested in understanding the structural, dynamic, and biophysical parameters that govern this recognition event, including designing altered peptide ligands (APLs) which seek to modulate the downstream signaling outcomes. However, dynamic links between modified peptide positions and distant residues have remained ill resolved until now. Using an integrative approach combining crystallographic ensemble and single models with atomistic molecular dynamics simulations and correlational analysis, we have established an approach that allows us to identify coupled dynamics between spatially distant residues at the pMHC interface. Furthermore, we constructed a network encoding the inter-residue couplings observed throughout the simulations. This computational workflow corroborates well with the functional and biophysical experimental data of our model system, and leads to novel insights regarding the differential immunogenicity of the closely related peptides analyzed in this study. Ultimately, we present an intuitive and comprehensive strategy for decoding the linked dynamics at the pMHC interface allowing for mechanistic insights into the biophysical bases governing immunogenicity.

BACKGROUND:TRIMELVax is a cancer vaccine prototype derived from heat-conditioned melanoma cell lysates combined with a natural adjuvant. Preclinical studies demonstrated robust antitumor immune responses and tumor regression. We conducted a Phase I clinical trial to evaluate the safety, tolerability, immunogenicity, and preliminary efficacy of TRIMELVax in patients with unresectable stage IV melanoma who had progressed after first-line anti-PD-1 therapy. METHODS:Eligible patients had stage IV melanoma with documented progression or unacceptable toxicity following anti-PD-1 treatment. TRIMELVax was administered subcutaneously every four weeks for a total of four doses. The primary endpoints of the study were safety and feasibility, which were assessed according to CTCAE v5.0. Secondary endpoints included efficacy as assessed by RECIST 1.1, overall survival (OS), progression-free survival (PFS), and immunogenicity, evaluated by peripheral blood immune cell responses and delayed-type hypersensitivity (DTH) reactions. RESULTS:Seventeen patients received ≥ 1 dose; 13 completed all four doses. Treatment-related adverse events occurred in 9 patients, with a grade 1 or 2 severity. Two patients experienced manageable grade 3 events. No grade 4-5 toxicities were observed. No complete responses were observed in this cohort. Notably, a partial response was observed in 1 patient, stable disease in 6 patients, yielding a 41% disease control rate; 10 patients progressed. Median OS was 14 months, and median PFS was 5.2 months. DTH positivity was observed in six of the nine patients tested, correlating with the induction of memory T cells in peripheral blood after treatment. Immunomonitoring revealed increased CXCR3 expression in CD8⁺ T cells and decreased CD39 expression in both CD4⁺ and CD8⁺ subsets in patients with disease control. One case with lung metastasis regression exhibited a significant expansion of TCF1⁺PD-1⁺ CD4⁺ and CD8⁺ T cell populations, and an enriched perforin⁺granzyme B⁺ CD8⁺ T cell compartment, consistent with vaccine-associated immune activation. CONCLUSIONS:TRIMELVax demonstrated acceptable safety and early signs of clinical activity in anti-PD-1 refractory melanoma patients. These findings support its immunogenic potential and warrant further evaluation in larger, controlled studies. CLINICALTRIAL:GOV: NCT06556004.

During direct communication between two cells, the plasma membranes of each cell serve as a platform for ligand-receptor interaction initiating downstream signaling cascades. In immune cell signaling, this cell-cell interface - the immune synapse - is highly spatiotemporally organized. Multiple stimulatory and co-stimulatory signals need to be integrated over time to ensure proper immune cell function. This process is still not fully understood given the vast complexity of interactions between proteins, lipids, glycocalyx and associated cortical actin cytoskeleton. Here, we presented a fully artificial model system to study the interface between two vesicles and a semi-artificial one between a live cell and a vesicle to reconstitute 3D contacts. We investigated the distribution and reorganization of immune cell proteins at artificial and semi-artificial contacts. We show the enrichment and depletion of different proteins in the synapse and how different peptides with varying affinity presented by the same MHC class I affect T cell activation. We further explored the distribution of glycocalyx elements and showed differential partitioning of different sugar moieties in the interface. While we focused on the T cell interface here, our model systems are powerful tools to study the distribution and reorganization of lipids, proteins and glycocalyx components at any cell-cell contact.

The precise recognition of specific peptide-major histocompatibility complex (pMHC) complexes by T cell receptors (TCRs) plays a key role in infectious disease, cancer, and autoimmunity. A critical step in many immunobiological studies is the identification of T cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in E. coli of H-2Db and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T cell repertoire in approaches ranging from yeast display to T cell staining.

Abstract Sinonasal cancer (SNC) is a rare and aggressive head and neck cancer with limited therapeutic options and an incompletely defined immune microenvironment. Although histopathology remains central to clinical tumor evaluation, immune profiling typically requires tissue dissociation and therefore loses the spatial relationships that shape antitumor immunity. Here, we combined single-cell RNA-sequencing approaches, spatial transcriptomics and Spatial V(D)J, a technique we recently pioneered, to analyse the cellular, spatial, and clonal architecture of SNC. We found that SNC tumors were marked by a robust T cell infiltration, including substantial proportions of classical tissue resident memory (TRM) and exhausted (TExh) CD8 + T cells. This inflamed phenotype was accompanied by an immense infiltration of suppressive regulatory T (Treg) cells, which differentiated towards a Tbet + CXCR3 + phenotype. Spatially, expanded Treg, CD8⁺ T and B cell clones concentrated within specialized peritumoral immune niches enriched for cancer-associated fibroblasts, CXCL9⁺ tumor-associated macrophages and LAMP3⁺IDO1⁺ dendritic cells. T cell clones occupying these niches were clonally related to those infiltrating epithelial tumor regions, linking lymphoid hubs to the broader tumor immune response. Together, these data identify SNC as an inflamed but highly immunoregulatory tumor type and reveal a spatially organized clonal architecture in which suppressive and cytotoxic lymphocyte states coexist within CXCL9⁺ myeloid niches.

Enhancing innate-adaptive immune crosstalk is key for improving cancer vaccine efficacy. TRIMELVax is a heat shock-conditioned whole-tumor-cell vaccine combining xenogeneic melanoma cell lysate, syngeneic B16F10 melanoma cell lysate, and Concholepas concholepas hemocyanin. Although TRIMELVax elicits robust antitumor responses in preclinical models, the mechanisms underlying its efficacy remain poorly defined. We characterized the early immune events triggered by TRIMELVax in mice using RT-qPCR, high-dimensional flow cytometry, immunohistochemistry, CFSE-based dendritic cell (DC) migration assays, and therapeutic melanoma models with transient neutrophil depletion. TRIMELVax elicited a rapid inflammatory response at the vaccination site, characterized by local upregulation of CXCL3, CXCL5, CXCL9, CCL3, CCL4, CCL12, IL-1β, IL-6/OSM, IL-12a, and G-CSF. This response drove an early influx of neutrophils and monocytes, followed by increased accumulation of cDC1, cDC2, and monocyte-derived DCs. Notably, we identified a transient population of neutrophils expressing markers associated with antigen-presenting cells (CD45⁺, CD11b⁺, Ly6G⁺, CD11c⁺, MHC-II⁺) that emerged within 12-24 hours postvaccination. These APC-like neutrophils colocalized with cDC1 at the injection site and subsequently migrated to the popliteal draining lymph nodes (pLN). Neutrophil depletion impaired cDC1 migration, reduced APC accumulation in pLN, and abolished the therapeutic efficacy of TRIMELVax. Together, these findings identify neutrophils as key early regulators of the innate inflammatory environment induced by TRIMELVax and suggest that neutrophils with APC-like features may impact DC trafficking and downstream antitumor immunity. Neutrophils, particularly those with APC-like phenotypes, emerge as promising cellular adjuvant targets for enhancing cancer vaccination strategies, offering a new avenue for rational vaccine design and combination with checkpoint blockade therapies.

The balance between affinity and specificity in T cell receptor (TCR)-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge for immunotherapy development. T cell engagers that circumvent these limitations are therefore of particular interest. We established a process to generate bispecific designed ankyrin repeat proteins (DARPins) that simultaneously target HLA-I/peptide complexes and CD3e. These high-affinity T cell engagers elicited CD8+ T cell activation against tumor targets with strong peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. A cryo-EM structure of the ternary DARPin/HLA-A∗0201/NY-ESO1157-165 complex revealed a rigid, concave DARPin surface spanning the full length of the peptide-binding cleft, contacting both α-helices and the peptide. The present findings reveal promising immuno-oncotherapeutic approaches and demonstrate the feasibility of rapidly developing DARPins with high affinity and specificity for HLA/peptide targets, which can be readily combined with a new generation of anti-CD3e-specific DARPins.

We report development and characterization of small non-immunoglobulin affibody affinity proteins directed to the highly glycosylated human carcinoembryonic antigen-related adhesion molecule 5 (CEACAM5, CEA), and their use in immunohistochemical (IHC) analyses of human pancreatic cancer samples and for in vivo tumor imaging. A total of nineteen unique anti-CEA affibodies were identified from large phage display libraries constructed using combinatorial protein engineering of a small 58 amino acid three-helix bundle protein domain. Molecular modeling suggested that all enriched clones share a binding surface with several clustered tryptophan residues interacting with a hydrophobic patch in the N1 domain of CEA centered around a phenylalanine residue. One variant, designated as C9, exhibited the highest affinity in biosensor analyses and was reformatted into a 15 kDa homodimer expressed in Escherichia coli. The biotinylated form, C9-C9-Bio, was evaluated for its IHC performance on matched frozen and formalin-fixed, paraffin-embedded (FFPE) sections of human pancreatic cancer samples (n = 7). Compared to clinical-grade monoclonal antibodies II-7 and CEA31, as well as a polyclonal reagent, C9-C9-Bio demonstrated highly sensitive CEA detection with minimal background staining. Statistical analyses including intraclass correlation and Bland-Altman assessments revealed excellent agreement between C9-C9-Bio and the two monoclonal antibodies in FFPE tissue samples. Further, a 99mTc[Tc]-labeled C9-C9 construct showed CEA-dependent binding to human cancer cell lines in vitro, and selectively bound to CEA-expressing BxPC3 xenografts in mice when investigated as a tracer for in vivo imaging, allowing for a visualization of tumors after four hours. In summary, these findings highlight the potential use of the easily produced CEA-binding C9 affibody for various clinical applications, including IHC and medical imaging, and as a targeting moiety for directing various therapeutic modalities to CEA-expressing tumors.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a central regulator of immune signalling, yet the conformational dynamics governing its activation remain poorly defined. Building on our earlier solution-state analysis of apo MALT1(PCASP-Ig3) 339–719 , which revealed domain flexibility, dynamic autoinhibition, and sensitivity to physiological ionic conditions, we combine NMR relaxation, molecular dynamics simulations, and ensemble modelling to delineate how solution environment reshapes its conformational landscape. Because most structural information derives from dimeric or inhibitor-bound states, the behaviour of monomeric, ligand-free MALT1 in physiological solution has remained unclear. Here, MD simulations show that low-salt conditions drive all trajectories toward a unified inactive-like ensemble, marked by inward rotation of W580 and coordinated rearrangements of Loop 2 and Loop 3, indicating that the inactive state is energetically favoured and its reactivation kinetically suppressed. Physiological ionic strength partially restores access to active-like loop motions, aligning with NMR evidence that sodium modulates catalytic readiness. In contrast, high-salt conditions rigidify the PCASP–Ig3 module, suppressing loop fluctuations and preventing active–inactive transitions, thereby strongly enriching the active-state population. Importantly, the combined MD–NMR analysis demonstrates that the NMR-initiated ensembles provide the most faithful representation of backbone and loop dynamics under low-salt conditions, capturing substrate-independent loop rearrangements, stable hydrophobic-core behaviour, and the intrinsic transitions that shape MALT1’s conformational equilibrium. Together, these findings identify ionic strength as a key regulator of MALT1 conformational equilibria,, highlighting how loop dynamics and domain flexibility tune its proteolytic competence and providing a dynamic framework for future structure-based modulation of MALT1 activity.

Se-methylselenocysteine (MSC) is a redox-active selenium-containing amino acid with notable anticancer potential, requiring enzymatic activation for cytotoxicity. Human kynurenine aminotransferase 1 (hKYAT1) catalyzes MSC through transamination and β-elimination pathways, generating β-methylselenopyruvate and methylselenol, both of which induce oxidative stress and epigenetic modulation. To enhance MSC metabolism and its therapeutic efficacy, we performed site-directed mutagenesis targeting three critical hKYAT1 residues: Tyr101, Asp126, and Phe278. These mutants, along with wild-type hKYAT1, were expressed in hepatocellular carcinoma cell lines HepG2 and Huh7, and their impact on enzymatic activity, cytotoxic effects, apoptosis and chromatin remodeling were evaluated. Several mutations significantly enhanced MSC metabolism, with Y101H and F278A increasing both transamination and β-elimination activity, and D126L favoring β-elimination. These modifications led to a five- to 30-fold increase in MSC-induced cytotoxicity compared to wild-type hKYAT1. Additionally, mutant hKYAT1 expression altered histone deacetylase (HDAC) profiles, increased histone H4 acetylation, and activated apoptotic signaling through caspase cleavage and cytochrome c release. Collectively, our findings demonstrate that rational engineering of hKYAT1 can potentiate MSC metabolism and amplify its anticancer effects, offering a promising enzyme-targeting strategy for selenium-based cancer therapies.

Stress granules (SGs) are cytoplasmic condensates of proteins and RNAs that form in response to cellular stress, playing a critical role in protecting cells during adverse conditions. G3BP proteins act as a central node and molecular switch in SG assembly. Growing evidence indicates that conformational changes in G3BP1 mediate its phase separation competence. However, the mechanistic basis underlying how G3BP conformational dynamics contribute to SG formation has remained elusive. Here, we demonstrate that the G3BP1 homodimer adopts both distinct expanded and compact conformations in solution, with transitions finely modulated by physiological changes in ionic strength and pH. Notably, the G3BP1(dRGG) deletion mutant, which is SG-incompetent, abolishes environmentally induced conformational transitions, underscoring the pivotal regulatory role of the RGG domain in G3BP1 dynamics. Additionally, G3BP1 can undergo RNA-independent phase separation at pH 6. Our findings establish a structural framework that connects the conformational plasticity of G3BP1 to its capacity for condensate assembly. We propose that the interplay between intrinsically disordered regions (IDRs), regulated by pH-driven conformational dynamics of G3BP1, finely tunes the threshold for initial granule assembly. These insights significantly enhance our understanding of SG dynamics and provide a mechanistic foundation for investigating how environmental stress conditions influence SG formation and function. ### Competing Interest Statement The authors have declared no competing interest.

Conformational heterogeneity is essential for protein function, yet validating theoretical molecular dynamics (MD) ensembles remains a significant challenge. In this study, we present an approach that integrates free MD simulations, starting from an AlphaFold-generated structure, with refined experimental NMR-relaxation data to identify biologically relevant holistic time-resolved 4D conformational ensembles. Specifically, we select trajectory segments (RMSD plateaus) consistent with experimental observables. For the extracellular region of Streptococcus pneumoniae PsrSp, we found that only specific segments of the long MD trajectory aligned well with experimental data. The resulting ensembles revealed two regions with increased flexibility, both of which play important functional roles.

OBJECTIVES:Anti-Jo1+ antisynthetase syndrome (ASyS) is characterised by autoantibodies targeting histidyl t-RNA synthetase (HisRS), association with HLA-DRB1*03:01 and a distinct clinical phenotype including interstitial lung disease, myositis, arthritis, and mechanic's hands. Previous studies of autoreactive HisRS-specific CD4+T cells point to yet undiscovered T cell epitopes. We aimed to identify new epitopes on HisRS to investigate the presence of autoreactive T cells and their corresponding T-cell receptor (TCR) repertoire from patients with ASyS. METHODS:Peptides from HisRS N-terminal region with appropriate major histocompatibility complex (MHC) anchor residues were selected for in vitro binding assays. The peptide (HisRS41-55) with the highest HLA-DRB1*03:01 binding affinity was selected for studies with HLA-class II tetramers. Peripheral blood mononuclear cells (PBMCs) from patients with ASyS with HLA-DRB1*03:01 (n = 12) were stimulated in vitro with peptide and peptide-HLA-DRB1*03:01 tetramers were used to detect HisRS+CD4+T cells. Single TCR sequencing of captured T cells allowed analyses of the underlying TCR repertoire. RESULTS:We identified a new T cell epitope on HisRS with high affinity for HLA-DRB1*03:01. Autoreactive HisRS+CD4+T cells were detected in PBMCs of patients (n = 6/12). TCR repertoire analysis of HisRS+CD4+T cells revealed shared gene V-alpha and beta usages. Moreover, HisRS+CD4+T cells persisted after treatment in 2 patients (P2 and P4) and 2 identical T cell clones were detected between the initial and follow-up time points in 1 patient (P2). CONCLUSIONS:Autoreactive T-cells targeting a new HisRS epitope were identified indicating T cell reactivity to diverse epitopes of the HisRS protein in patients with anti-Jo1 autoantibodies. Furthermore, we demonstrated the TCR repertoire of autoreactive HisRS+CD4+T cells in patients. Persistence of these T-cells and specific clones may be contributing to disease.

The interaction between a class I peptide-major histocompatibility complex (pMHC) and a T cell receptor (TCR) plays a central role in the elicitation of CD8+ T cell immune responses. As a result, considerable effort has been invested in understanding the structural, dynamic, and biophysical parameters that govern this recognition event, including designing altered peptide ligands (APLs) which seek to modulate the downstream signaling outcomes. However, dynamic links between modified peptide positions and distant residues have until yet been ill resolved. Using an integrative approach combining crystallographic ensemble and single models with atomistic molecular dynamics simulations and correlational analysis, we have established an approach that allows us to identify coupled dynamics between spatially distant residues at the pMHC interface. Furthermore, we constructed a network encoding the inter-residue couplings observed throughout the simulations. This computational workflow corroborates well with experimental data and leads to novel insights regarding the differential immunogenicity of the closely related peptides analyzed in this study. Ultimately, we present an intuitive and comprehensive strategy for decoding the linked dynamics at the pMHC interface allowing for mechanistic insights into the biophysical bases governing immunogenicity. One Sentence Summary The dynamics at the pMHC interface can be encoded as a biophysically relevant network to yield molecular insights into immunogenicity ### Competing Interest Statement The authors have declared no competing interest. Insamlingsstiftelsen Cancer- och Allergifonden, 10399 Cancerfonden, 24 3775 Pj 01 H Vetenskapsrådet, 2021-05061 Stiftelsen Konung Gustaf V:s Jubileumsfond, 244092

Human kynurenine aminotransferase 1 (hKYAT1) plays a crucial role in the transamination of aromatic amino acids and kynurenine. This promiscuous homodimeric enzyme transaminates various amino acids into their corresponding α-keto acids. Additionally, hKYAT1 is known to catalyze the β-elimination of cysteine-S conjugates and cysteine-Se conjugates. In this study, we performed mutational analyses of hKYAT1, targeting its catalytic, ligand-binding, and substrate-binding sites. The transamination activity of 13 mutant variants was systematically evaluated against sixteen different amino acid substrates, including kynurenine, selenomethionine (SeMet), and Se-methylselenocysteine (MSC), as well as for the β-elimination of SeMet and MSC. Our results demonstrate that mutations of residues E27 in the catalytic site and H279 in the substratestabilizing site significantly enhanced the transamination of several amino acids, including phenylalanine, tryptophan, histidine, and MSC. The H279F mutation increased transamination and β-elimination of MSC by 2- and 1.5-fold, respectively. Furthermore, mutation at the ligand-binding residues R398, F125, and N185 substantially reduced MSC transamination activity of hKYAT1. Interestingly, none of the tested mutations affected the transamination of l-kynurenine, a natural substrate of hKYAT1. Altogether, these findings support future investigation into hKYAT1 as a modifiable target in selenium-mediated anticancer approaches.

The crystal structure of the extracellular region of the second pneumococcal LCP, a polyisoprenyl-teichoic acid-peptidoglycan teichoic acid transferase PsrSp, was determined and refined to 2.15 Å resolution. Despite the low sequence homology with other LCP proteins, the PsrSp maintains the fold of the LCP domain, and the positions of the residues suggested to participate in the transferase function are conserved. The tunnel found in the PsrSp between the central β-sheet and three α-helices is wide enough to accommodate polyisoprenyl-teichoic acid. Comparison of the crystallographic temperature factors of LCP from distinct bacteria demonstrated that the four long loops located close to the teichoic acid and peptidoglycan binding sites have different relative mobilities. To compare the dynamics of the PsrSp in crystalline state and in solution, NMR spectra were recorded, and 88% of the residues were assigned in the 1H-15N TROSY HSQC spectra. Perfect accordance in the secondary structure of the crystal structure of PsrSp with NMR data demonstrated correct assignment. Moreover, the relative mobility of the essential loops estimated from the crystallographic B-factor is in good agreement with order parameter S2, predicted from chemical shift. We hypothesize that the dynamics of these loops are important for the substrate promiscuity of LCP proteins.