Loss-of-function cancer-linked mutations in the EIF4G2 non-canonical translation initiation factor.

Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here, we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions and, importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near-complete loss of function. Two other mutations within the MIF4G domain specifically affected EIF4G2's ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects.

A pathogenic variant in the uncharacterized RNF212B gene results in severe aneuploidy male infertility and repeated IVF failure.

Quantitative and qualitative spermatogenic impairments are major causes of men's infertility. Although in vitro fertilization (IVF) is effective, some couples persistently fail to conceive. To identify causal variants in patients with severe male infertility factor and repeated IVF failures, we sequenced the exome of two consanguineous family members who underwent several failed IVF cycles and were diagnosed with low sperm count and motility. We identified a rare homozygous nonsense mutation in a previously uncharacterized gene, RNF212B, as the causative variant. Recurrence was identified in another unrelated, infertile patient who also faced repeated failed IVF treatments. scRNA-seq demonstrated meiosis-specific expression of RNF212B. Sequence analysis located a protein domain known to be associated with aneuploidy, which can explain multiple IVF failures. Accordingly, FISH analysis revealed a high aneuploidy rate in the patients' sperm cells and their IVF embryos. Finally, inactivation of the Drosophila orthologs significantly reduced male fertility. Given that members of the evolutionary conserved RNF212 gene family are involved in meiotic recombination and crossover maturation, our findings indicate a critical role of RNF212B in meiosis, genome stability, and in human fertility. Since recombination is completely absent in Drosophila males, our findings may indicate an additional unrelated role for the RNF212-like paralogs in spermatogenesis.

Structural and biochemical analysis of a novel atypically split intein reveals a conserved histidine specific to cysteine-less inteins.

Protein trans-splicing mediated by a split intein reconstitutes a protein backbone from two parts. This virtually traceless autoprocessive reaction provides the basis for numerous protein engineering applications. Protein splicing typically proceeds through two thioester or oxyester intermediates involving the side chains of cysteine or serine/threonine residues. A cysteine-less split intein has recently attracted particular interest as it can splice under oxidizing conditions and is orthogonal to disulfide or thiol bioconjugation chemistries. Here, we report the split PolB16 OarG intein, a second such cysteine-independent intein. As a unique trait, it is atypically split with a short intein-N precursor fragment of only 15 amino acids, the shortest characterized to date, which was chemically synthesized to enable protein semi-synthesis. By rational engineering we obtained a high-yielding, improved split intein mutant. Structural and mutational analysis revealed the dispensability of the usually crucial conserved motif N3 (block B) histidine as an obvious peculiar property. Unexpectedly, we identified a previously unnoticed histidine in hydrogen-bond forming distance to the catalytic serine 1 as critical for splicing. This histidine has been overlooked so far in multiple sequence alignments and is highly conserved only in cysteine-independent inteins as a part of a newly discovered motif NX. The motif NX histidine is thus likely of general importance to the specialized environment in the active site required in this intein subgroup. Together, our study advances the toolbox as well as the structural and mechanistic understanding of cysteine-less inteins.

Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis.

Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein.

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of non-redundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions.

Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody-epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.

Correction: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides.

[This corrects the article DOI: 10.18632/oncotarget.10516.].

A new MEIOB mutation is a recurrent cause for azoospermia and testicular meiotic arrest.

STUDY QUESTION: Are there genetic variants that can be used for the clinical evaluation of azoospermic men? SUMMARY ANSWER: A novel homozygous frame-shift mutation in the MEIOB gene was identified in three azoospermic patients from two different families. WHAT IS KNOWN ALREADY: Up to 1% of all men have complete absence of sperm in the semen, a condition known as azoospermia. There are very few tools for determining the etiology of azoospermia and the likelihood of sperm cells in the testis. The MEIOB gene codes for a single-strand DNA binding protein required for DNA double-strand breaks repair during meiosis. MEIOB appears to be exclusively expressed in human and mouse testis, and MeioB knockout mice are azoospermic due to meiotic arrest. STUDY DESIGN, SIZE, DURATION: Two brothers with non-obstructive azoospermia (NOA) underwent whole-exome sequencing followed by comprehensive bioinformatics analyses. Candidate variations were further screened in infertile and fertile men, as well as in public and local reference databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 159 infertile and 77 fertile men. The exomes of two Arab men were completely sequenced. In addition, 213 other men of the same Arab ethnicity (136 infertile and 77 fertile men) underwent restriction fragment length polymorphism (RFLP) screening, as did 21 NOA men, of other ethnicities, with testicular impairment of spermatocyte arrest. All of the infertile men underwent Y-chromosome microdeletion and CFTR gene mutation assessments. Comprehensive bioinformatics analyses were designed to uncover candidate mutations associated with azoospermia. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous frame-shift mutation in the MEIOB gene was identified in two brothers of Arab ethnicity. This frame-shift is predicted to result in a truncated MEIOB protein, which lacks the conserved C-terminal DNA binding domain. RFLP screening of the mutation in 157 infertile men, including 112 NOA patients of Arab ethnicity, identified an additional unrelated NOA patient with the same homozygous mutation and a similar testicular impairment. This mutation was not found in available public databases (n > 160 000), nor in the 77 proven fertile men, nor in our database of local Israeli population variations derived from exome and genome sequencing data (n = 500). LIMITATIONS, REASONS FOR CAUTION: We have thus far screened for only two specific MEIOB probable pathogenic mutations in a relatively small local cohort. Therefore, the relative incidence of MEIOB mutations in azoospermia should be further assessed in larger and diverse cohorts in order to determine the efficiency of MEIOB sequence screening for clinical evaluations. WIDER IMPLICATIONS OF THE FINDINGS: The relatively high incidence of likely NOA-causing mutations in MEIOB that was found in our cohort supports the idea that a complete screening of this gene might be beneficial for clinical evaluation of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This research was supported in part by a grant to EA from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC grant agreement (616088). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A cancer associated somatic mutation in LC3B attenuates its binding to E1-like ATG7 protein and subsequent lipidation.

Macroautophagy/autophagy is a conserved catabolic process that maintains cellular homeostasis under basal growth and stress conditions. In cancer, autophagy can either prevent or promote tumor growth, at early or advanced stages, respectively. We screened public databases to identify autophagy-related somatic mutations in cancer, using a computational approach to identify cancer mutational target sites, employing exact statistics. The top significant hit was a missense mutation (Y113C) in the MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) protein, which occurred at a significant frequency in cancer, and was detected in early stages in primary tumors of patients with known tumor lineage. The mutation reduced the formation of GFP-LC3B puncta and attenuated LC3B lipidation during Torin1-induced autophagy. Its effect on the direct physical interaction of LC3B with each of the 4 proteins that control its maturation or lipidation was tested by applying a protein-fragment complementation assay and co-immunoprecipitation experiments. Interactions with ATG4A and ATG4B proteases were reduced, yet without perturbing the cleavage of mutant LC3B. Most importantly, the mutation significantly reduced the interaction with the E1-like enzyme ATG7, but not the direct interaction with the E2-like enzyme ATG3, suggesting a selective perturbation in the binding of LC3B to some of its partner proteins. Structure analysis and molecular dynamics simulations of LC3B protein and its mutant suggest that the mutation changes the conformation of a loop that has several contact sites with ATG4B and the ATG7 homodimer. We suggest that this loss-of-function mutation, which attenuates autophagy, may promote early stages of cancer development.

Identification and Characterization of Differentially-Regulated Type IVb Pilin Genes Necessary for Predation in Obligate Bacterial Predators.

Bdellovibrio bacteriovorus is an obligate predator of bacteria that grows and divides within the periplasm of its prey. Functions involved in the early steps of predation have been identified and characterized, but mediators of prey invasion are still poorly detailed. By combining omics data available for Bdellovibrio and like organisms (BALO's), we identified 43 genes expressed in B. bacteriovorus during the early interaction with prey. These included genes in a tight adherence (TAD) operon encoding for two type IVb fimbriae-like pilin proteins (flp1 and flp2), and their processing and export machinery. Two additional flp genes (flp3 and flp4) were computationally identified at other locations along the chromosome, defining the largest and most diverse type IVb complement known in bacteria to date. Only flp1, flp2 and flp4 were expressed; their respective gene knock-outs resulted in a complete loss of the predatory ability without losing the ability to adhere to prey cells. Additionally, we further demonstrate differential regulation of the flp genes as the TAD operon of BALOs with different predatory strategies is controlled by a flagellar sigma factor FliA, while flp4 is not. Finally, we show that FliA, a known flagellar transcriptional regulator in other bacteria, is an essential Bdellovibrio gene.

A familial study of azoospermic men identifies three novel causative mutations in three new human azoospermia genes.

PURPOSE: Up to 1% of all men experience azoospermia, a condition of complete absence of sperm in the semen. The mechanisms and genes involved in spermatogenesis are mainly studied in model organisms, and their relevance to humans is unclear because human genetic studies are very scarce. Our objective was to uncover novel human mutations and genes causing azoospermia due to testicular meiotic maturation arrest. METHODS: Affected and unaffected siblings from three families were subjected to whole-exome or whole-genome sequencing, followed by comprehensive bioinformatics analyses to identify mutations suspected to cause azoospermia. These likely mutations were further screened in azoospermic and normozoospermic men and in men proven to be fertile, as well as in a reference database of local populations. RESULTS: We identified three novel likely causative mutations of azoospermia in three genes: MEIOB, TEX14, and DNAH6. These genes are associated with different meiotic processes: meiotic crossovers, daughter cell abscission, and possibly rapid prophase movements. CONCLUSION: The genes and pathways we identified are fundamental for delineating common causes of azoospermia originating in mutations affecting diverse meiotic processes and have great potential for accelerating approaches to diagnose, treat, and prevent infertility.Genet Med advance online publication 16 February 2017.

The landscape of sex-differential transcriptome and its consequent selection in human adults.

BACKGROUND: The prevalence of several human morbid phenotypes is sometimes much higher than intuitively expected. This can directly arise from the presence of two sexes, male and female, in one species. Men and women have almost identical genomes but are distinctly dimorphic, with dissimilar disease susceptibilities. Sexually dimorphic traits mainly result from differential expression of genes present in both sexes. Such genes can be subject to different, and even opposing, selection constraints in the two sexes. This can impact human evolution by differential selection on mutations with dissimilar effects on the two sexes. RESULTS: We comprehensively mapped human sex-differential genetic architecture across 53 tissues. Analyzing available RNA-sequencing data from 544 adults revealed thousands of genes differentially expressed in the reproductive tracts and tissues common to both sexes. Sex-differential genes are related to various biological systems, and suggest new insights into the pathophysiology of diverse human diseases. We also identified a significant association between sex-specific gene transcription and reduced selection efficiency and accumulation of deleterious mutations, which might affect the prevalence of different traits and diseases. Interestingly, many of the sex-specific genes that also undergo reduced selection efficiency are essential for successful reproduction in men or women. This seeming paradox might partially explain the high incidence of human infertility. CONCLUSIONS: This work provides a comprehensive overview of the sex-differential transcriptome and its importance to human evolution and human physiology in health and in disease.

Development of a screening system for inteins active in protein splicing based on intein insertion into the LacZα-peptide.

Protein splicing by inteins has found diverse applications in biotechnology, protein chemistry and chemical biology. Inteins display a wide range of efficiencies and rates unpredictable from their amino acid sequences. Here, we identified positions T22S and S35 in the LacZα peptide as intein insertion sites that strictly require protein splicing, in contrast to cleavage side-reactions, to allow for complementation of ß-galactosidase activity. Both the cis-variant of the M86 mutant of the Ssp DnaB intein and a split form undergoing protein trans-splicing gave rise to formation of blue colonies in the ß-galactosidase read-out. Furthermore, we report the two novel, naturally split VidaL T4Lh-1 and VidaL UvsX-2 inteins whose N-terminal fragments consist of only 15 and 16 amino acids, respectively. Initial biochemical characterization with the LacZα host system of these inteins further underlines its utility. Finally, we used the LacZα host system to rapidly identify amino acid substitutions from a small randomized library at the structurally conserved intein position 2 next to the catalytic center, that are tolerated for protein splicing activity of the M86 intein. These findings demonstrate the potential of the system for initial testing and directed evolution of inteins.

Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides.

The p53 tumor suppressor serves as a major barrier against malignant transformation. Over 50% of tumors inactivate p53 by point mutations in its DNA binding domain. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, misfolded form. The equilibrium between the properly folded and misfolded states of p53 may be affected by molecules that interact with p53, stabilizing its native folding and restoring wild type p53 activity to cancer cells. To select for mutant p53 (mutp53) reactivating peptides, we adopted the phage display technology, allowing interactions between mutp53 and random peptide libraries presented on phages and enriching for phage that favor the correctly folded p53 conformation. We obtained a large database of potential reactivating peptides. Lead peptides were synthesized and analyzed for their ability to restore proper p53 folding and activity. Remarkably, many enriched peptides corresponded to known p53-binding proteins, including RAD9. Importantly, lead peptides elicited dramatic regression of aggressive tumors in mouse xenograft models. Such peptides might serve as novel agents for human cancer therapy.

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus.

UNLABELLED: Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. IMPORTANCE: In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Cell-cycle progress in obligate predatory bacteria is dependent upon sequential sensing of prey recognition and prey quality cues.

Predators feed on prey to acquire the nutrients necessary to sustain their survival, growth, and replication. In Bdellovibrio bacteriovorus, an obligate predator of Gram-negative bacteria, cell growth and replication are tied to a shift from a motile, free-living phase of search and attack to a sessile, intracellular phase of growth and replication during which a single prey cell is consumed. Engagement and sustenance of growth are achieved through the sensing of two unidentified prey-derived cues. We developed a novel ex vivo cultivation system for B. bacteriovorus composed of prey ghost cells that are recognized and invaded by the predator. By manipulating their content, we demonstrated that an early cue is located in the prey envelope and a late cue is found within the prey soluble fraction. These spatially and temporally separated cues elicit discrete and combinatory regulatory effects on gene transcription. Together, they delimit a poorly characterized transitory phase between the attack phase and the growth phase, during which the bdelloplast (the invaded prey cell) is constructed. This transitory phase constitutes a checkpoint in which the late cue presumably acts as a determinant of the prey's nutritional value before the predator commits. These regulatory adaptations to a unique bacterial lifestyle have not been reported previously.

Absence of α- and ß-dystroglycan is associated with Walker-Warburg syndrome.

OBJECTIVE: To identify the underlying genetic defect in 5 patients from a consanguineous family with a Walker-Warburg phenotype, together with intracranial calcifications. METHODS: Homozygosity mapping and exome sequencing, followed by Sanger sequencing of the obtained candidate gene, was performed. Expression of the candidate gene was tested by reverse transcription PCR. Patient fibroblasts were converted to myotubes, and the expression and function of dystroglycan was tested by Western blotting. RESULTS: We detected a homozygous loss-of-function frameshift mutation in the DAG1 gene and showed that this mutation results in a complete absence of both α- and ß-dystroglycan. CONCLUSIONS: A loss-of-function mutation in DAG1 can result in Walker-Warburg syndrome and is not embryonic lethal.

Reduced selection and accumulation of deleterious mutations in genes exclusively expressed in men.

Sex-limited selection can moderate the elimination of deleterious mutations from the population and contribute to the high prevalence of common human diseases. Accordingly, deleterious mutations in autosomal genes that are exclusively expressed in only one of the sexes undergo sex-limited selection and can reach higher frequencies than mutations similarly selected in both sexes. Here we show that the number of deleterious SNPs in genes exclusively expressed in men is twofold higher than in genes that are selected in both sexes. Additional analyses suggest that the increased number of damaging mutations we found in male-specific genes is due to reduced selection in females. These results are noteworthy since many of these male-specific genes are known to be crucial for male reproduction, and are thus likely to be under strong purifying selection. We suggest that inheritance of male-infertility-causative mutations through unaffected female lineages contributes to the high incidence of male infertility.

An atypical naturally split intein engineered for highly efficient protein labeling.

Protein trans-splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL-TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N-terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans-splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50-fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi-synthesis and other intein-related biotechnological applications.

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 µm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.